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Role of the Endocannabinoid System in the Regulation of Intestinal Homeostasis

  • Hailey Cuddihey
    Affiliations
    Snyder Institute for Chronic Diseases, University of Calgary’s Cumming School of Medicine, Calgary, Alberta, Canada

    Hotchkiss Brain Institute, University of Calgary, Calgary, Alberta, Canada

    Department of Physiology and Pharmacology, Cumming School of Medicine, University of Calgary, Calgary, Alberta, Canada
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  • Wallace K. MacNaughton
    Affiliations
    Snyder Institute for Chronic Diseases, University of Calgary’s Cumming School of Medicine, Calgary, Alberta, Canada

    Alberta Children’s Hospital Research Institute, University of Calgary, Calgary, Alberta, Canada

    Inflammation Research Network, University of Calgary, Calgary, Alberta, Canada

    Department of Physiology and Pharmacology, Cumming School of Medicine, University of Calgary, Calgary, Alberta, Canada
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  • Keith A. Sharkey
    Correspondence
    Correspondence Address correspondence to: Keith Sharkey, PhD, Department of Physiology and Pharmacology, Cumming School of Medicine, University of Calgary, 3330 Hospital Drive NW, Calgary, AB T2N 4N1, Canada. fax: XXX.
    Affiliations
    Snyder Institute for Chronic Diseases, University of Calgary’s Cumming School of Medicine, Calgary, Alberta, Canada

    Hotchkiss Brain Institute, University of Calgary, Calgary, Alberta, Canada

    Department of Physiology and Pharmacology, Cumming School of Medicine, University of Calgary, Calgary, Alberta, Canada
    Search for articles by this author
Open AccessPublished:June 21, 2022DOI:https://doi.org/10.1016/j.jcmgh.2022.05.015
      The maintenance of intestinal homeostasis is fundamentally important to health. Intestinal barrier function and immune regulation are key determinants of intestinal homeostasis and are therefore tightly regulated by a variety of signaling mechanisms. The endocannabinoid system is a lipid mediator signaling system widely expressed in the gastrointestinal tract. Accumulating evidence suggests the endocannabinoid system is a critical nexus involved in the physiological processes that underlie the control of intestinal homeostasis. In this review we will illustrate how the endocannabinoid system is involved in regulation of intestinal permeability, fluid secretion, and immune regulation. We will also demonstrate a reciprocal regulation between the endocannabinoid system and the gut microbiome. The role of the endocannabinoid system is complex and multifaceted, responding to both internal and external factors while also serving as an effector system for the maintenance of intestinal homeostasis.

      Keywords

      Abbreviations used in this paper:

      2-AG (2-arachidonoylglycerol), 2-OG (2-oleoylglycerol), 2-PG (2-palmitoylglycerol), ABHD (α/β-hydrolase domain-containing protein), AEA (anandamide), cAMP (cyclic adenosine monophosphate), CB (cannabinoid), CBD (cannabidiol), CNS (central nervous system), DAGL (diacylglycerol lipase), DIO (diet-induced obese), DSS (dextran sodium sulfate), ECS (endocannabinoid system), ENS (enteric nervous system), FAAH (fatty acid amide hydrolase), GI (gastrointestinal), GPCR (G protein-coupled receptor), IBD (inflammatory bowel disease), MAGL (monoacylglycerol lipase), NAAA (N-acylethanolamine-hydrolyzing acid amidase), NAE (N-acylethanolamide), NAPE (N-arachidonoyl phosphatidylethanolamine), OEA (N-oleoylethanolamide), PEA (N-palmitoylethanolamide), PLC (phospholipase C), PLD (phospholipase D), PPAR (peroxisome proliferator-activated receptor), TEER (transepithelial electrical resistance), THC (Δ9-tetrahydrocannabinol), TNBS (trinitrobenzene sulfonic acid), TRPV1 (transient receptor potential vanilloid type 1), ZO (zonula occludens)
      The endocannabinoid system is a lipid mediator signaling system widely distributed throughout the gastrointestinal tract. The endocannabinoid system plays a pivotal role in the maintenance of intestinal homeostasis and gut barrier integrity, responding to internal and external environmental factors while also serving as a homeostatic effector system.
      The maintenance of intestinal homeostasis is of fundamental importance to health. Intestinal homeostasis requires the integration of digestive and defensive functions of the gut to protect against the insults that arise from digestion, harmful pathogens and toxins, and the commensal microbiota that live in the gut, while simultaneously promoting the efficient utilization of food. A central determinant of intestinal homeostasis is intestinal barrier function.
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      Figure thumbnail gr1
      Figure 1Endocannabinoid synthesis and degradation pathways. Synthesis and degradation of the 2 major endocannabinoids, anandamide (A) and 2-AG (B). Further details are provided in the text. 2-AG, 2-arachidonoylglycerol; DAGL, diacylglycerol lipase; FAAH, fatty acid amide hydrolase; GDE1, glycerophosphodiesterase E1; MAGL, monoacylglycerol lipase; NAPE-PLD, N-acylphosphatidylethanolamine-hydrolyzing phospholipase D; PI, phosphatidylinositol; PLA, phospholipase A; PLC, phospholipase C; PLD, phospholipase D; PTPN22, protein tyrosine phosphatase non-receptor type 22. Figure created with BioRender.com.
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      Figure thumbnail gr2
      Figure 2Signaling pathways induced by endocannabinoid receptor activation. Anandamide is produced from N-arachidonoyl phosphatidylethanolamine (NAPE) by the action of N-acylphosphatidylethanolamine-hydrolyzing phospholipase D (NAPE-PLD). It is degraded by fatty acid amide hydrolase (FAAH). FAAH hydrolyzes AEA into arachidonic acid (AA) and ethanolamine (EA). 2-Arachidonoylglycerol (2-AG) is primarily produced through the action of diacylglycerol lipase (DAGL). It is degraded by monoacylglycerol lipase into AA and glycerol. Inhibition of these degradative enzymes increases levels of endocannabinoid to enhance signaling. Both anandamide and 2-AG signal through the CB1 and CB2 receptors where anandamide is a partial agonist (light arrows) and 2-AG is a full agonist (thick arrows). Upon ligand binding, both receptors activate Gαi/o, which then interacts with β and γ subunits to initiate downstream signaling. The primary response is inhibition of adenylate cyclase (AC) and therefore reduction in the cytosolic levels of cyclic adenosine monophosphate (cAMP). There is evidence suggesting that these receptors can also activate MAP kinase (MAPK) pathways including ERK, JNK, and p38MAPK. Activation of the CB1 receptor also triggers β-arrestin, which is involved in receptor internalization, desensitization, and degradation, and which may also be involved in intracellular signaling pathways. Figure created with BioRender.com.
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      • Duncan M.
      • Mouihate A.
      • Mackie K.
      • Keenan C.M.
      • Buckley N.E.
      • Davison J.S.
      • Patel K.D.
      • Pittman Q.J.
      • Sharkey K.A.
      Cannabinoid CB 2 receptors in the enteric nervous system modulate gastrointestinal contractility in lipopolysaccharide-treated rats.
      • Wright K.
      • Rooney N.
      • Feeney M.
      • Tate J.
      • Robertson D.
      • Welham M.
      • Ward S.
      Differential expression of cannabinoid receptors in the human colon: cannabinoids promote epithelial wound healing.
      • Galiègue S.
      • Mary S.
      • Marchand J.
      • Dussossoy D.
      • Carrière D.
      • Carayon P.
      • Bouaboula M.
      • Shire D.
      • Le Fur G.
      • Casellas P.
      Expression of central and peripheral cannabinoid receptors in human immune tissues and leukocyte subpopulations.
      • Beltramo M.
      • Bernardini N.
      • Bertorelli R.
      • Campanella M.
      • Nicolussi E.
      • Fredduzzi S.
      • Reggiani A.
      CB2 receptor-mediated antihyperalgesia: possible direct involvement of neural mechanisms.
      • Cabral G.A.
      • Staab A.
      Effects on the immune system.
      Like CB1, activation of CB2 inhibits the release of immune mediators or neurotransmitters.
      To rapidly activate their receptors, endocannabinoids, as lipids, require a carrier protein to overcome the hydrophilic environment of the extracellular space or synapse. Fatty acid-binding proteins, albumin, and heat shock protein 70 have been described as endocannabinoid carrier proteins.
      • Oddi S.
      • Fezza F.
      • Pasquariello N.
      • D’Agostino A.
      • Catanzaro G.
      • De Simone C.
      • Rapino C.
      • Finazzi-Agrò A.
      • Maccarrone M.
      Molecular identification of albumin and Hsp70 as cytosolic anandamide-binding proteins.
      • Kaczocha M.
      • Glaser S.T.
      • Deutsch D.G.
      Identification of intracellular carriers for the endocannabinoid anandamide.
      • Kaczocha M.
      • Vivieca S.
      • Sun J.
      • Glaser S.T.
      • Deutsch D.G.
      Fatty acid-binding proteins transport N-acylethanolamines to nuclear receptors and are targets of endocannabinoid transport inhibitors.
      Fatty acid-binding proteins were discovered as mediators of the intracellular transport of AEA from the plasma membrane for degradation by FAAH.
      • Kaczocha M.
      • Glaser S.T.
      • Deutsch D.G.
      Identification of intracellular carriers for the endocannabinoid anandamide.
      Whether they are used by AEA extracellularly remains uncertain. Fatty acid-binding protein 5 was found to regulate 2-AG signaling at excitatory glutamatergic synapses in the brain.
      • Haj-Dahmane S.
      • Shen R.Y.
      • Elmes M.W.
      • Studholme K.
      • Kanjiya M.P.
      • Bogdan D.
      • Thanos P.K.
      • Miyauchi J.T.
      • Tsirka S.E.
      • Deutsch D.G.
      • Kaczocha M.
      Fatty-acid-binding protein 5 controls retrograde endocannabinoid signaling at central glutamate synapses.
      In vitro evidence was that the source of the fatty acid-binding protein 5 was astrocytes,
      • Haj-Dahmane S.
      • Shen R.Y.
      • Elmes M.W.
      • Studholme K.
      • Kanjiya M.P.
      • Bogdan D.
      • Thanos P.K.
      • Miyauchi J.T.
      • Tsirka S.E.
      • Deutsch D.G.
      • Kaczocha M.
      Fatty-acid-binding protein 5 controls retrograde endocannabinoid signaling at central glutamate synapses.
      which suggests that fatty acid-binding proteins are extracellular carrier proteins. These data suggest that glial cells serve to limit the extent of synaptic endocannabinoid signaling. Whether this mechanism exists in vivo remains to be determined.
      Recent single cell RNA sequencing of neurons and glia in the ENS reveal fatty acid-binding protein 5 gene is highly expressed by enteric neurons, enteroendocrine cells, and also in enteric glia.
      • Zeisel A.
      • Hochgerner H.
      • Lönnerberg P.
      • Johnsson A.
      • Memic F.
      • van der Zwan J.
      • Häring M.
      • Braun E.
      • Borm L.E.
      • La Manno G.
      • Codeluppi S.
      • Furlan A.
      • Lee K.
      • Skene N.
      • Harris K.D.
      • Hjerling-Leffler J.
      • Arenas E.
      • Ernfors P.
      • Marklund U.
      • Linnarsson S.
      Molecular architecture of the mouse nervous system.
      ,
      • Drokhlyansky E.
      • Smillie C.S.
      • Van Wittenberghe N.
      • Ericsson M.
      • Griffin G.K.
      • Eraslan G.
      • Dionne D.
      • Cuoco M.S.
      • Goder-Reiser M.N.
      • Sharova T.
      • Kuksenko O.
      • Aguirre A.J.
      • Boland G.M.
      • Graham D.
      • Rozenblatt-Rosen O.
      • Xavier R.J.
      • Regev A.
      The human and mouse enteric nervous system at single-cell resolution.
      What role it plays in the ENS remains to be determined. It also remains to be determined how endocannabinoid signaling between other cell types in the gut, eg, immune cells, occurs in vivo.
      The ECS is not restricted to the actions of its 2 primary ligands. Several related bioactive lipids have been identified that structurally resemble AEA and 2-AG. These include the N-acylethanolamides (NAEs), N-palmitoylethanolamide (PEA), N-oleoylethanolamide (OEA), N-stearoylethanolamide, and N-linoleoylethanolamide, as well as lipid mediators from the acylglycerol family, 2-palmitoylglycerol (2-PG) and 2-oleoylglycerol (2-OG). These bioactive lipids are thought to modulate endocannabinoid signaling through interactions with synthetic and degradative enzymes and other non-canonical endocannabinoid receptors.
      • Cristino L.
      • Bisogno T.
      • Di Marzo V.
      Cannabinoids and the expanded endocannabinoid system in neurological disorders.
      ,
      • Hansen H.S.
      • Vana V.
      Non-endocannabinoid N-acylethanolamines and 2-monoacylglycerols in the intestine.
      ,
      • Tsuboi K.
      • Uyama T.
      • Okamoto Y.
      • Ueda N.
      Endocannabinoids and related N-acylethanolamines: biological activities and metabolism.
      In addition, endocannabinoids do not interact exclusively with the canonical CB receptors, but also with several other classes of receptors.
      • Pertwee R.G.
      • Howlett A.C.
      • Abood M.E.
      • Alexander S.P.H.
      • Di Marzo V.
      • Elphick M.R.
      • Greasley P.J.
      • Hansen H.S.
      • Kunos G.
      • Mackie K.
      • Mechoulam R.
      • Ross R.A.
      International union of basic and clinical pharmacology: LXXIX—cannabinoid receptors and their ligands: beyond CB1 and CB2.
      One of the best examples of endocannabinoid action at a non-cannabinoid receptor is the affinity of AEA for the transient receptor potential vanilloid type-1 (TRPV1) channel.
      • Pertwee R.G.
      • Howlett A.C.
      • Abood M.E.
      • Alexander S.P.H.
      • Di Marzo V.
      • Elphick M.R.
      • Greasley P.J.
      • Hansen H.S.
      • Kunos G.
      • Mackie K.
      • Mechoulam R.
      • Ross R.A.
      International union of basic and clinical pharmacology: LXXIX—cannabinoid receptors and their ligands: beyond CB1 and CB2.
      Endocannabinoids and NAEs also have affinity for peroxisome proliferator-activated receptor (PPAR)-α, PPAR-γ, G protein-coupled receptor 55 (GPR55), GPR119, and GPR18.
      • Cani P.D.
      • Plovier H.
      • Van Hul M.
      • Geurts L.
      • Delzenne N.M.
      • Druart C.
      • Everard A.
      Endocannabinoids: at the crossroads between the gut microbiota and host metabolism.
      ,
      • Ryberg E.
      • Larsson N.
      • Sjögren S.
      • Hjorth S.
      • Hermansson N.-O.
      • Leonova J.
      • Elebring T.
      • Nilsson K.
      • Drmota T.
      • Greasley P.J.
      The orphan receptor GPR55 is a novel cannabinoid receptor.
      • Kohno M.
      • Hasegawa H.
      • Inoue A.
      • Muraoka M.
      • Miyazaki T.
      • Oka K.
      • Yasukawa M.
      Identification of N-arachidonylglycine as the endogenous ligand for orphan G-protein-coupled receptor GPR18.
      • De Petrocellis L.
      • Di Marzo V.
      Non-CB1, non-CB2 receptors for endocannabinoids, plant cannabinoids, and synthetic cannabimimetics: focus on G-protein-coupled receptors and transient receptor potential channels.
      • Syed S.K.
      • Hoang Bui H.
      • Beavers L.S.
      • Farb T.B.
      • Ficorilli J.
      • Chesterfield A.K.
      • Kuo M.-S.
      • Bokvist K.
      • Barrett D.G.
      • Efanov A.M.
      Regulation of GPR119 receptor activity with endocannabinoid-like lipids.
      Thus, the ECS consists of lipid mediators, their associated biosynthetic and degradative enzymes, receptors, and intracellular signaling systems capable of exquisite local regulatory control in physiological and pathophysiological conditions. There remains much we do not know about this system in the gut, notably the exact sites and specific conditions in which endocannabinoids are released. We know little about the genetic and epigenetic regulation of CB receptor expression in the GI tract, or that of other components of the ECS in the gut. Moreover, we know relatively little about how the ECS is regulated or dysregulated in pathophysiological conditions, although we have evidence for alterations in the expression of the components of the ECS in various GI diseases.
      In the following sections of this review, we will discuss how the ECS is involved in the control of intestinal homeostasis by considering the elements that make up the physical, secretory, and immunologic components of the intestinal barrier (Figure 3). Finally, we will consider the reciprocal regulation of the ECS and the gut microbiome.
      Figure thumbnail gr3
      Figure 3Endocannabinoid system (ECS) interactions with barrier, immune, and microbiome systems. The ECS can alter the barrier function of the epithelium by increasing insertion of tight junction (TJ) proteins to enhance the physical barrier of the epithelium. Endocannabinoids also decrease chloride secretion to decrease the secretory barrier. The ECS, primarily through activation of CB2 receptors, decreases immune cell function by decreasing cytokine release. There are reciprocal interactions between the ECS and the microbiome. By affecting each of these systems, the ECS therefore also regulates the complex crosstalk that exists between the epithelium and the immune system, the immune system and the microbiome, and the microbiome and the epithelium. Figure created with BioRender.com.

      Regulation of Epithelial Tight Junctions by the Endocannabinoid System

      A single layer of epithelial cells lines the GI tract and is all that separates the contents of the gut from the rest of the body.
      • Furness J.B.
      • Rivera L.R.
      • Cho H.-J.
      • Bravo D.M.
      • Callaghan B.
      The gut as a sensory organ.
      The intestinal epithelium is a selectively permeable barrier that facilitates the transcellular and paracellular transport of nutrients, water, and electrolytes, enables antigen sampling for oral tolerance, while inhibiting the passage of possibly harmful substances such as toxins, foreign antigens, pathogens, and commensal bacteria.
      • Lee S.H.
      Intestinal permeability regulation by tight junction: implication on inflammatory bowel diseases.
      ,
      • Loh G.
      • Blaut M.
      Role of commensal gut bacteria in inflammatory bowel diseases.
      Paracellular transport across the epithelial barrier is regulated by tight junctions. Tight junctions are protein complexes located between adjacent epithelial cells that form a selectively permeable barrier.
      • Nusrat A.
      • Turner J.R.
      • Madara J.L.
      Molecular physiology and pathophysiology of tight junctions: IV—regulation of tight junctions by extracellular stimuli: nutrients, cytokines, and immune cells.
      There are 4 groups of transmembrane proteins: occluding,
      • Furuse M.
      • Hirase T.
      • Itoh M.
      • Nagafuchi A.
      • Yonemura S.
      • Tsukita S.
      • Tsukita S.
      Occludin: a novel integral membrane protein localizing at tight junctions.
      claudins,
      • Furuse M.
      • Fujita K.
      • Hiiragi T.
      • Fujimoto K.
      • Tsukita S.
      Claudin-1 and -2: novel integral membrane proteins localizing at tight junctions with no sequence similarity to occludin.
      junctional adhesion molecule,
      • Martìn-Padura I.
      • Lostaglio S.
      • Schneemann M.
      • Williams L.
      • Romano M.
      • Fruscella P.
      • Panzeri C.
      • Stoppacciaro A.
      • Ruco L.
      • Villa A.
      • Simmons D.
      • Dejana E.
      Junctional adhesion molecule, a novel member of the immunoglobulin superfamily that distributes at intercellular junctions and modulates monocyte transmigration.
      and tricellulin.
      • Ikenouchi J.
      • Furuse M.
      • Furuse K.
      • Sasaki H.
      • Tsukita S.
      • Tsukita S.
      Tricellulin constitutes a novel barrier at tricellular contacts of epithelial cells.
      Scaffold proteins, such as zonula occludens (ZO) proteins, anchor the intracellular domains of the transmembrane proteins to the actin cytoskeleton.
      • Lee S.H.
      Intestinal permeability regulation by tight junction: implication on inflammatory bowel diseases.
      The regulation of tight junctions is dynamic and controls the paracellular permeability of ions, nutrients, and water.
      • Lee S.H.
      Intestinal permeability regulation by tight junction: implication on inflammatory bowel diseases.
      ,
      • Nusrat A.
      • Turner J.R.
      • Madara J.L.
      Molecular physiology and pathophysiology of tight junctions: IV—regulation of tight junctions by extracellular stimuli: nutrients, cytokines, and immune cells.
      When the integrity of the tight junction barrier is compromised, paracellular permeability increases and allows the passage of large molecules and microbes that reside in the gut lumen. This can lead to inflammation and tissue damage from the infiltration of bacteria and their metabolites into the mucosa.
      • Lee S.H.
      Intestinal permeability regulation by tight junction: implication on inflammatory bowel diseases.
      The intestinal epithelium expresses CB receptors, although this varies according to species, location along the GI tract, and pathophysiological state. The human colon expresses CB1 receptors with what appears to be primarily apical expression based on immunohistochemical studies.
      • Wright K.
      • Rooney N.
      • Feeney M.
      • Tate J.
      • Robertson D.
      • Welham M.
      • Ward S.
      Differential expression of cannabinoid receptors in the human colon: cannabinoids promote epithelial wound healing.
      Colonic epithelia also express CB2 receptors in Crohn’s disease. The epithelium of mouse small intestine and colon do not display a substantial degree of CB1 expression under normal conditions.
      • Casu M.A.
      • Porcella A.
      • Ruiu S.
      • Saba P.
      • Marchese G.
      • Carai M.A.M.
      • Reali R.
      • Gessa G.L.
      • Pani L.
      Differential distribution of functional cannabinoid CB1 receptors in the mouse gastroenteric tract.
      ,
      • Grill M.
      • Hasenoehrl C.
      • Kienzl M.
      • Kargl J.
      • Schicho R.
      Cellular localization and regulation of receptors and enzymes of the endocannabinoid system in intestinal and systemic inflammation.
      Caco-2 cells, a cancer cell line often used to study epithelial cell biology, expresses CB1 receptors.
      • Karwad M.A.
      • Couch D.G.
      • Theophilidou E.
      • Sarmad S.
      • Barrett D.A.
      • Larvin M.
      • Wright K.L.
      • Lund J.N.
      • O’Sullivan S.E.
      The role of CB1 in intestinal permeability and inflammation.
      The ECS has been shown to play a role in the regulation of tight junction proteins essential for the maintenance of intestinal barrier function.
      • Karwad M.A.
      • Couch D.G.
      • Theophilidou E.
      • Sarmad S.
      • Barrett D.A.
      • Larvin M.
      • Wright K.L.
      • Lund J.N.
      • O’Sullivan S.E.
      The role of CB1 in intestinal permeability and inflammation.
      • Muccioli G.G.
      • Naslain D.
      • Bäckhed F.
      • Reigstad C.S.
      • Lambert D.M.
      • Delzenne N.M.
      • Cani P.D.
      The endocannabinoid system links gut microbiota to adipogenesis.
      • Alhamoruni A.
      • Lee A.C.
      • Wright K.L.
      • Larvin M.
      • O’Sullivan S.E.
      Pharmacological effects of cannabinoids on the Caco-2 cell culture model of intestinal permeability.
      In an in vitro model of the intestinal epithelium using Caco-2 cell monolayers, Muccioli et al
      • Muccioli G.G.
      • Naslain D.
      • Bäckhed F.
      • Reigstad C.S.
      • Lambert D.M.
      • Delzenne N.M.
      • Cani P.D.
      The endocannabinoid system links gut microbiota to adipogenesis.
      demonstrated that simultaneous application of the CB1 agonist HU210 and lipopolysaccharide reduced transepithelial electrical resistance (TEER), and this was associated with a reduction in the expression of tight junction proteins occludin and ZO-1. The effect on TEER and tight junction protein expression was blocked by the CB1 antagonist rimonabant.
      • Muccioli G.G.
      • Naslain D.
      • Bäckhed F.
      • Reigstad C.S.
      • Lambert D.M.
      • Delzenne N.M.
      • Cani P.D.
      The endocannabinoid system links gut microbiota to adipogenesis.
      Similarly, Alhamoruni et al
      • Alhamoruni A.
      • Lee A.C.
      • Wright K.L.
      • Larvin M.
      • O’Sullivan S.E.
      Pharmacological effects of cannabinoids on the Caco-2 cell culture model of intestinal permeability.
      showed that apical application of the endocannabinoids AEA and 2-AG reduced TEER, increased mRNA expression of ZO-1, and reduced mRNA expression of claudin-1. This effect was also blocked by the CB1 antagonist AM251, suggesting that ligands of the ECS modulate intestinal permeability under baseline conditions by altering the expression of tight junction proteins.
      Moreover, the ECS has also been shown to play a role in the regulation of intestinal permeability in conditions where baseline intestinal permeability is perturbed.
      • Karwad M.A.
      • Couch D.G.
      • Theophilidou E.
      • Sarmad S.
      • Barrett D.A.
      • Larvin M.
      • Wright K.L.
      • Lund J.N.
      • O’Sullivan S.E.
      The role of CB1 in intestinal permeability and inflammation.
      ,
      • Alhamoruni A.
      • Lee A.C.
      • Wright K.L.
      • Larvin M.
      • O’Sullivan S.E.
      Pharmacological effects of cannabinoids on the Caco-2 cell culture model of intestinal permeability.
      • Wang J.
      • Zhang X.
      • Yang C.
      • Zhao S.
      Effect of monoacylglycerol lipase inhibition on intestinal permeability in chronic stress model.
      • Alhamoruni A.
      • Wright K.L.
      • Larvin M.
      • O’Sullivan S.E.
      Cannabinoids mediate opposing effects on inflammation-induced intestinal permeability.
      In an in vitro model of ethylenediamine tetraacetic acid–induced increased permeability, basolateral application of AEA and 2-AG decreased permeability in Caco-2 cell monolayers.
      • Alhamoruni A.
      • Lee A.C.
      • Wright K.L.
      • Larvin M.
      • O’Sullivan S.E.
      Pharmacological effects of cannabinoids on the Caco-2 cell culture model of intestinal permeability.
      Although the effect of AEA and 2-AG is mediated by CB1, AEA relied on the additional recruitment of TRPV1.
      • Alhamoruni A.
      • Lee A.C.
      • Wright K.L.
      • Larvin M.
      • O’Sullivan S.E.
      Pharmacological effects of cannabinoids on the Caco-2 cell culture model of intestinal permeability.
      In a model of inflammation-induced increase in permeability where Caco-2 cells are exposed to tumor necrosis factor and interferon gamma, basolateral application of AEA and 2-AG had no effect on permeability.
      • Alhamoruni A.
      • Wright K.L.
      • Larvin M.
      • O’Sullivan S.E.
      Cannabinoids mediate opposing effects on inflammation-induced intestinal permeability.
      However, in both models, apical application of AEA and 2-AG increased permeability through a CB1-dependent mechanism.
      • Alhamoruni A.
      • Lee A.C.
      • Wright K.L.
      • Larvin M.
      • O’Sullivan S.E.
      Pharmacological effects of cannabinoids on the Caco-2 cell culture model of intestinal permeability.
      ,
      • Alhamoruni A.
      • Wright K.L.
      • Larvin M.
      • O’Sullivan S.E.
      Cannabinoids mediate opposing effects on inflammation-induced intestinal permeability.
      Apical application of the phytocannabinoids THC and CBD were protective and reduced permeability through a CB1-dependent mechanism.
      • Alhamoruni A.
      • Lee A.C.
      • Wright K.L.
      • Larvin M.
      • O’Sullivan S.E.
      Pharmacological effects of cannabinoids on the Caco-2 cell culture model of intestinal permeability.
      ,
      • Alhamoruni A.
      • Wright K.L.
      • Larvin M.
      • O’Sullivan S.E.
      Cannabinoids mediate opposing effects on inflammation-induced intestinal permeability.
      Interestingly, co-application of PEA to the basolateral compartment and cannabidiol to the apical compartment decreased permeability, and this was dependent on PPARα and CB1, respectively.
      • Karwad M.A.
      • Couch D.G.
      • Theophilidou E.
      • Sarmad S.
      • Barrett D.A.
      • Larvin M.
      • Wright K.L.
      • Lund J.N.
      • O’Sullivan S.E.
      The role of CB1 in intestinal permeability and inflammation.
      Altogether, these data demonstrate that the ECS is involved in the regulation of gut barrier function largely through a CB1-dependent mechanism but may also rely on the recruitment of TRPV1 and PPARα.
      Additional studies in vivo have further supported the role of the ECS in the regulation of intestinal permeability. Chronic activation of the CB1 receptor with the potent full agonist HU210 in wild-type mice led to an increase in permeability to 4 kDa FITC-dextran.
      • Muccioli G.G.
      • Naslain D.
      • Bäckhed F.
      • Reigstad C.S.
      • Lambert D.M.
      • Delzenne N.M.
      • Cani P.D.
      The endocannabinoid system links gut microbiota to adipogenesis.
      In contrast, Zoppi et al
      • Zoppi S.
      • Madrigal J.L.M.
      • Pérez-Nievas B.G.
      • Marín-Jiménez I.
      • Caso J.R.
      • Alou L.
      • García-Bueno B.
      • Colón A.
      • Manzanares J.
      • Luisa Gómez-Lus M.
      • Menchén L.
      • Leza J.C.
      Endogenous cannabinoid system regulates intestinal barrier function in vivo through cannabinoid type 1 receptor activation.
      demonstrated that CB1 knockout mice experience a greater degree of intestinal barrier dysfunction after exposure to immobilization and acoustic stress compared with wild-type mice, suggesting that the CB1 receptor exerts a protective role in the colon in the regulation of paracellular permeability. Consistent with these findings, Chen et al
      • Chen M.
      • Hou P.
      • Zhou M.
      • Ren Q.
      • Wang X.
      • Huang L.
      • Hui S.
      • Yi L.
      • Mi M.
      Resveratrol attenuates high-fat diet-induced non-alcoholic steatohepatitis by maintaining gut barrier integrity and inhibiting gut inflammation through regulation of the endocannabinoid system.
      showed that the beneficial effect of resveratrol on intestinal permeability in a high-fat diet–induced nonalcoholic steatohepatitis model in rats was blocked by the synthetic CB1 receptor agonist arachidonyl-2´-chloroethylamide. Interestingly, more recent work demonstrated that CB1 receptor agonists and antagonists reduce intestinal permeability in mice exposed to a 2-week high-fat diet. Although changes in the expression of claudin-2 may underlie the effect of the CB1 agonist, further studies are required to understand the mechanism by which the CB1 antagonist reduces intestinal permeability.

      Cuddihey H, Cavin JB, Wallace LE, Vemuri K, Makriyannis A, MacNaughton WK, Sharkey KA. Role of CB1 receptors in the acute regulation of small intestinal permeability: effects of high-fat diet. Am J Physiol Gastrointest Liver Physiol.

      Obesity is a metabolic disorder associated with an altered gut microbiota, defects in barrier function, and increased endocannabinoid tone. In genetically obese (Ob/Ob) mice and dietary-induced obese mice, treatment with the CB1 antagonist rimonabant reduced plasma lipopolysaccharide levels and led to a change in the distribution and localization of tight junction proteins ZO-1 and occludin, suggesting a decrease in intestinal permeability.
      • Muccioli G.G.
      • Naslain D.
      • Bäckhed F.
      • Reigstad C.S.
      • Lambert D.M.
      • Delzenne N.M.
      • Cani P.D.
      The endocannabinoid system links gut microbiota to adipogenesis.
      ,
      • Mehrpouya-Bahrami P.
      • Chitrala K.N.
      • Ganewatta M.S.
      • Tang C.
      • Murphy E.A.
      • Enos R.T.
      • Velazquez K.T.
      • McCellan J.
      • Nagarkatti M.
      • Nagarkatti P.
      Blockade of CB1 cannabinoid receptor alters gut microbiota and attenuates inflammation and diet-induced obesity.
      Together, these data suggest that the ECS is indeed involved in the chronic regulation of intestinal permeability in vivo through a CB1-dependent mechanism. CB1 agonists disrupt the gut barrier and, as suggested by Cani et al,
      • Cani P.D.
      • Plovier H.
      • Van Hul M.
      • Geurts L.
      • Delzenne N.M.
      • Druart C.
      • Everard A.
      Endocannabinoids: at the crossroads between the gut microbiota and host metabolism.
      act as a gate opener (increasing intestinal permeability), whereas CB1 antagonists protect the gut barrier and are considered gate keepers. This CB1-mediated disruption of intestinal permeability could be explained partially by changes in the distribution and localization of tight junction proteins after CB1 receptor activation. CB1 receptor blockade restores the tight junction barrier and the integrity of the gut barrier. However, in recent studies, we found that CB1 agonists could also reduce intestinal permeability in vitro under baseline conditions,

      Cuddihey H, Cavin JB, Wallace LE, Vemuri K, Makriyannis A, MacNaughton WK, Sharkey KA. Role of CB1 receptors in the acute regulation of small intestinal permeability: effects of high-fat diet. Am J Physiol Gastrointest Liver Physiol.

      suggesting that the pharmacologic regulation of CB1 receptors may depend on the level of endocannabinoid tone or other factors.
      The literature has suggested that AEA and 2-AG, two endogenous CB1 agonists, exert differential effects on gut barrier integrity, acting themselves as a gate opener and gate keeper, respectively. Although the literature consistently establishes the role of AEA as a gate opener, the evidence to support 2-AG as a gate keeper is rather limited. In vitro data have suggested that AEA and 2-AG exert the same effects on barrier function such that they increase permeability when applied to the apical compartment and decrease permeability when applied to the basolateral compartment of Caco-2 cell monolayers. However, in vivo data have indirectly suggested a role for 2-AG as a gate keeper. Administration of Akkermansia muciniphila in mice with diet-induced obesity (DIO) is associated with an increase in 2-AG and an improvement of gut barrier function. From this study, the authors suggested that 2-AG is indeed a gate keeper, although the data to support this idea are quite limited. The role of 2-AG as a gate keeper is somewhat challenging to understand because 2-AG is a full CB1 agonist and typically produces effects associated with CB1 agonism.
      • Reggio P.
      Endocannabinoid binding to the cannabinoid receptors: what is known and what remains unknown.
      If 2-AG is indeed a gate keeper, then this CB1 agonist is behaving more like an antagonist, because CB1 antagonists have been shown to decrease permeability. On the other hand, AEA is a partial CB1 agonist with lower efficacy than 2-AG
      • Cristino L.
      • Becker T.
      • Di Marzo V.
      Endocannabinoids and energy homeostasis: an update.
      but is considered a gate opener and is therefore behaving like a CB1 agonist. On the basis of this information, one might predict the opposite of what has been shown, such that 2-AG would be the gate opener and AEA would be the gate keeper. Whether 2-AG is indeed a gate keeper remains unclear and warrants further investigation. Further studies will need to address the concept of biased agonism in CB1-mediated effects on epithelial permeability; AEA and 2-AG, while both acting at epithelially expressed CB1 receptor, could activate different signaling pathways resulting in different cellular responses. In addition, it is not known whether differences in the polarization of distribution of CB1 receptor, apical vs basolateral, could be associated with coupling to different signaling pathways and, hence, different effects on epithelial cell function.

      Regulation of Secretory Function by the Endocannabinoid System

      The secretion of fluid, mucus, antimicrobial peptides, and secretory immunoglobulin A are key elements of intestinal barrier function.
      • Wells J.M.
      • Brummer R.J.
      • Derrien M.
      • MacDonald T.T.
      • Troost F.
      • Cani P.D.
      • Theodorou V.
      • Dekker J.
      • Méheust A.
      • De Vos W.M.
      • Mercenier A.
      • Nauta A.
      • Garcia-Rodenas C.L.
      Homeostasis of the gut barrier and potential biomarkers.
      ,
      • Vancamelbeke M.
      • Vermeire S.
      The intestinal barrier: a fundamental role in health and disease.
      • Pietrzak B.
      • Tomela K.
      • Olejnik-Schmidt A.
      • Mackiewicz A.
      • Schmidt M.
      Secretory IgA in intestinal mucosal secretions as an adaptive barrier against microbial cells.
      • Bevins C.L.
      • Salzman N.H.
      Paneth cells, antimicrobial peptides and maintenance of intestinal homeostasis.
      The role of the ECS in the control of intestinal mucus, antimicrobial peptide, and secretory immunoglobulin A secretion is not well-understood. However, the ECS is involved in the regulation of neurogenic intestinal fluid secretion under baseline conditions
      • MacNaughton W.K.
      • Van Sickle M.D.
      • Keenan C.M.
      • Cushing K.
      • Mackie K.
      • Sharkey K.A.
      Distribution and function of the cannabinoid-1 receptor in the modulation of ion transport in the guinea pig ileum: relationship to capsaicin-sensitive nerves.
      • Tyler K.
      • Hillard C.J.
      • Greenwood-Van Meerveld B.
      Inhibition of small intestinal secretion by cannabinoids is CB1 receptor-mediated in rats.
      • Wasilewski A.
      • Sacharczuk M.
      • Fichna J.
      Modulation of the endocannabinoid system by the fatty acid amide hydrolase, monoacylglycerol and diacylglycerol lipase inhibitors as an attractive target for secretory diarrhoea therapy.
      and when fluid secretion is enhanced to respond to luminal toxin. Oral administration of cholera toxin to mice results in a large increase in fluid accumulation in the small intestine.
      • Izzo A.A.
      • Capasso F.
      • Costagliola A.
      • Bisogno T.
      • Marsicano G.
      • Ligresti A.
      • Matias I.
      • Capasso R.
      • Pinto L.
      • Borrelli F.
      • Cecio A.
      • Lutz B.
      • Mascolo N.
      • Di Marzo V.
      An endogenous cannabinoid tone attenuates cholera toxin-induced fluid accumulation in mice.
      This is associated with increased levels of AEA and increased expression of the CB1 receptor mRNA. These responses are involved in the homeostatic control because endogenous CB1 receptor agonists reduce secretion to control levels, and a CB1 receptor antagonist further exacerbates secretion.
      • Izzo A.A.
      • Capasso F.
      • Costagliola A.
      • Bisogno T.
      • Marsicano G.
      • Ligresti A.
      • Matias I.
      • Capasso R.
      • Pinto L.
      • Borrelli F.
      • Cecio A.
      • Lutz B.
      • Mascolo N.
      • Di Marzo V.
      An endogenous cannabinoid tone attenuates cholera toxin-induced fluid accumulation in mice.
      In in vitro preparations of guinea pig ileum, CB1 agonism seems to reduce chloride ion transport (the main driver of fluid secretion) through prevention of neurotransmitter release from primary sensory afferents, not through direct effects on the epithelium.
      • MacNaughton W.K.
      • Van Sickle M.D.
      • Keenan C.M.
      • Cushing K.
      • Mackie K.
      • Sharkey K.A.
      Distribution and function of the cannabinoid-1 receptor in the modulation of ion transport in the guinea pig ileum: relationship to capsaicin-sensitive nerves.
      There is limited evidence supporting a role for CB2 receptors in the regulation of intestinal secretion.
      • Wasilewski A.
      • Sacharczuk M.
      • Fichna J.
      Modulation of the endocannabinoid system by the fatty acid amide hydrolase, monoacylglycerol and diacylglycerol lipase inhibitors as an attractive target for secretory diarrhoea therapy.

      Regulation of Local Gastrointestinal Immune Function by Endocannabinoids

      The ECS is an important regulator of the immune system.
      • Rahaman O.
      • Ganguly D.
      Endocannabinoids in immune regulation and immunopathologies.
      • Cabral G.A.
      • Ferreira G.A.
      • Jamerson M.J.
      Endocannabinoids and the immune system in health and disease.
      • Pandey R.
      • Mousawy K.
      • Nagarkatti M.
      • Nagarkatti P.
      Endocannabinoids and immune regulation.
      Of note, cells of the innate and adaptive immune systems express CB2 receptors that control their activity. Within the GI tract, neutrophils, macrophages, and T and B cells express CB2 receptor as well as other receptors of the endocannabinoidome (eg, PPARα and GPR55).
      • Grill M.
      • Hasenoehrl C.
      • Kienzl M.
      • Kargl J.
      • Schicho R.
      Cellular localization and regulation of receptors and enzymes of the endocannabinoid system in intestinal and systemic inflammation.
      ,
      • Szabady R.L.
      • Louissaint C.
      • Lubben A.
      • Xie B.
      • Reeksting S.
      • Tuohy C.
      • Demma Z.
      • Foley S.E.
      • Faherty C.S.
      • Llanos-Chea A.
      • Olive A.J.
      • Mrsny R.J.
      • McCormick B.A.
      Intestinal P-glycoprotein exports endocannabinoids to prevent inflammation and maintain homeostasis.
      • Acharya N.
      • Penukonda S.
      • Shcheglova T.
      • Hagymasi A.T.
      • Basu S.
      • Srivastava P.K.
      Endocannabinoid system acts as a regulator of immune homeostasis in the gut.
      • Gentili M.
      • Ronchetti S.
      • Ricci E.
      • Di Paola R.
      • Gugliandolo E.
      • Cuzzocrea S.
      • Bereshchenko O.
      • Migliorati G.
      • Riccardi C.
      Selective CB2 inverse agonist JTE907 drives T cell differentiation towards a Treg cell phenotype and ameliorates inflammation in a mouse model of inflammatory bowel disease.
      • Leinwand K.L.
      • Jones A.A.
      • Huang R.H.
      • Jedlicka P.
      • Kao D.J.
      • de Zoeten E.F.
      • Ghosh S.
      • Moaddel R.
      • Wehkamp J.
      • Ostaff M.J.
      • Bader J.
      • Aherne C.M.
      • Collins C.B.
      Cannabinoid receptor-2 ameliorates inflammation in murine model of Crohn’s disease.
      • Ziring D.
      • Wei B.
      • Velazquez P.
      • Schrage M.
      • Buckley N.E.
      • Braun J.
      Formation of B and T cell subsets require the cannabinoid receptor CB2.
      The ECS acts as a regulator of immune homeostasis in the gut. For example, CB2 receptor regulates the numbers of CX3CR1Hi macrophages in the intestinal lamina propria and their tolerogenic potential.
      • Acharya N.
      • Penukonda S.
      • Shcheglova T.
      • Hagymasi A.T.
      • Basu S.
      • Srivastava P.K.
      Endocannabinoid system acts as a regulator of immune homeostasis in the gut.
      Similarly, activation of CB2 enhances the expansion of regulatory T cells in the gut with concomitant anti-inflammatory actions.
      • Acharya N.
      • Penukonda S.
      • Shcheglova T.
      • Hagymasi A.T.
      • Basu S.
      • Srivastava P.K.
      Endocannabinoid system acts as a regulator of immune homeostasis in the gut.
      • Gentili M.
      • Ronchetti S.
      • Ricci E.
      • Di Paola R.
      • Gugliandolo E.
      • Cuzzocrea S.
      • Bereshchenko O.
      • Migliorati G.
      • Riccardi C.
      Selective CB2 inverse agonist JTE907 drives T cell differentiation towards a Treg cell phenotype and ameliorates inflammation in a mouse model of inflammatory bowel disease.
      • Leinwand K.L.
      • Jones A.A.
      • Huang R.H.
      • Jedlicka P.
      • Kao D.J.
      • de Zoeten E.F.
      • Ghosh S.
      • Moaddel R.
      • Wehkamp J.
      • Ostaff M.J.
      • Bader J.
      • Aherne C.M.
      • Collins C.B.
      Cannabinoid receptor-2 ameliorates inflammation in murine model of Crohn’s disease.
      • Ziring D.
      • Wei B.
      • Velazquez P.
      • Schrage M.
      • Buckley N.E.
      • Braun J.
      Formation of B and T cell subsets require the cannabinoid receptor CB2.
      In the section below we will expand on these homeostatic actions by focusing on the role of the ECS in regulating intestinal homeostasis in response to bacterial pathogens and intestinal inflammation.

      Role of the Endocannabinoid System in Regulating Intestinal Homeostasis in Response to Bacterial Pathogens and Intestinal Inflammation

      One of the first lines of defense against invading pathogens is the recruitment of neutrophils to the site of infection. The rapid transepithelial migration of neutrophils is a critical response to combat enteric infection. However, the products of neutrophil degranulation and the oxidative burst that are used to kill bacteria are associated with significant damage to surrounding tissues that if unchecked can lead to tissue damage and dysfunction.
      • Nusrat A.
      • Parkos C.A.
      • Liang T.W.
      • Carnes D.K.
      • Madara J.L.
      Neutrophil migration across model intestinal epithelia: monolayer disruption and subsequent events in epithelial repair.
      There are various lipid mediators involved in the resolution of inflammation and tissue restitution including the resolvins and lipoxin A4,
      • Gewirtz A.T.
      • Collier-Hyams L.S.
      • Young A.N.
      • Kucharzik T.
      • Guilford W.J.
      • Parkinson J.F.
      • Williams I.R.
      • Neish A.S.
      • Madara J.L.
      Lipoxin A 4 analogs attenuate induction of intestinal epithelial proinflammatory gene expression and reduce the severity of dextran sodium sulfate-induced colitis.
      ,
      • Serhan C.N.
      • Levy B.D.
      Resolvins in inflammation: emergence of the pro-resolving superfamily of mediators.
      but recently, endocannabinoids were discovered as endogenous regulators of the proinflammatory actions of the eicosanoid hepoxilin A3. Szabady et al
      • Szabady R.L.
      • Louissaint C.
      • Lubben A.
      • Xie B.
      • Reeksting S.
      • Tuohy C.
      • Demma Z.
      • Foley S.E.
      • Faherty C.S.
      • Llanos-Chea A.
      • Olive A.J.
      • Mrsny R.J.
      • McCormick B.A.
      Intestinal P-glycoprotein exports endocannabinoids to prevent inflammation and maintain homeostasis.
      investigated the role of the epithelial P-glycoprotein efflux pump in countering the hepoxilin A3 proinflammatory pathway. They discovered that N-acylethanolamines (AEA, OEA, and α-linolenoylethanolamide), effluxed via P-glycoprotein, suppressed neutrophil migration mediated by hepoxilin A3. This effect was sensitive to FAAH, but not MAGL, and was shown to be mediated by the CB2 receptor localized on neutrophils. Interestingly, Szabady et al did not find evidence for a role for 2-AG or its congeners in inhibiting neutrophil migration, despite the fact it is a full CB2 receptor agonist.
      • Szabady R.L.
      • Louissaint C.
      • Lubben A.
      • Xie B.
      • Reeksting S.
      • Tuohy C.
      • Demma Z.
      • Foley S.E.
      • Faherty C.S.
      • Llanos-Chea A.
      • Olive A.J.
      • Mrsny R.J.
      • McCormick B.A.
      Intestinal P-glycoprotein exports endocannabinoids to prevent inflammation and maintain homeostasis.
      Thus, although AEA is a (relatively weak) CB2 agonist, exactly how other NAEs regulate CB2 receptor function and the specific receptor mechanisms responsible for these effects remain to be explained.
      Consistent with these data, CB2 receptor knockout mice treated with either dextran sodium sulfate (DSS) or trinitrobenzene sulfonic acid (TNBS) to induce colitis have more severe disease and dramatic increases in neutrophil accumulation in the intestinal lumen.
      • Szabady R.L.
      • Louissaint C.
      • Lubben A.
      • Xie B.
      • Reeksting S.
      • Tuohy C.
      • Demma Z.
      • Foley S.E.
      • Faherty C.S.
      • Llanos-Chea A.
      • Olive A.J.
      • Mrsny R.J.
      • McCormick B.A.
      Intestinal P-glycoprotein exports endocannabinoids to prevent inflammation and maintain homeostasis.
      ,
      • Engel M.A.
      • Kellerman C.A.
      • Burnat G.
      • Hahn E.G.
      • Rau T.
      • Konturek P.C.
      Mice lacking cannabinoid CB1-, CB2-receptors or both receptors show increased susceptibility to trinitrobenzene sulfonic acid (TNBS)-induced colitis.
      Similarly, activation of CB2 receptors directly with specific agonists or indirectly by inhibiting FAAH and elevating endogenous ligands protects against experimental colitis in mice.
      • Gentili M.
      • Ronchetti S.
      • Ricci E.
      • Di Paola R.
      • Gugliandolo E.
      • Cuzzocrea S.
      • Bereshchenko O.
      • Migliorati G.
      • Riccardi C.
      Selective CB2 inverse agonist JTE907 drives T cell differentiation towards a Treg cell phenotype and ameliorates inflammation in a mouse model of inflammatory bowel disease.
      ,
      • D’Argenio G.
      • Valenti M.
      • Scaglione G.
      • Cosenza V.
      • Sorrentini I.
      • Di Marzo V.
      Up-regulation of anandamide levels as an endogenous mechanism and a pharmacological strategy to limit colon inflammation.
      • Storr M.A.
      • Keenan C.M.
      • Zhang H.
      • Patel K.D.
      • Makriyannis A.
      • Sharkey K.A.
      Activation of the cannabinoid 2 receptor (CB2) protects against experimental colitis.
      • Storr M.A.
      • Keenan C.M.
      • Emmerdinger D.
      • Zhang H.
      • Yüce B.
      • Sibaev A.
      • Massa F.
      • Buckley N.E.
      • Lutz B.
      • Göke B.
      • Brand S.
      • Patel K.D.
      • Sharkey K.A.
      Targeting endocannabinoid degradation protects against experimental colitis in mice: involvement of CB1 and CB2 receptors.
      • Sałaga M.
      • Mokrowiecka A.
      • Zakrzewski P.K.
      • Cygankiewicz A.
      • Leishman E.
      • Sobczak M.
      • Zatorski H.
      • Małecka-Panas E.
      • Kordek R.
      • Storr M.
      • Krajewska W.M.
      • Bradshaw H.B.
      • Fichna J.
      Experimental colitis in mice is attenuated by changes in the levels of endocannabinoid metabolites induced by selective inhibition of fatty acid amide hydrolase (FAAH).
      In accordance with these findings, the CB2-Q63R variant contributes to the risk for pediatric IBD, particularly Crohn’s disease,
      • Strisciuglio C.
      • Bellini G.
      • Miele E.
      • Martinelli M.
      • Cenni S.
      • Tortora C.
      • Tolone C.
      • Miraglia Del Giudice E.
      • Rossi F.
      Cannabinoid receptor 2 functional variant contributes to the risk for pediatric inflammatory bowel disease.
      illustrating the clinical significance of endocannabinoid receptor mechanisms to intestinal homeostasis. In fact, the endocannabinoidome is markedly dysregulated in IBD,
      • Grill M.
      • Högenauer C.
      • Blesl A.
      • Haybaeck J.
      • Golob-Schwarzl N.
      • Ferreirós N.
      • Thomas D.
      • Gurke R.
      • Trötzmüller M.
      • Köfeler H.C.
      • Gallé B.
      • Schicho R.
      Members of the endocannabinoid system are distinctly regulated in inflammatory bowel disease and colorectal cancer.
      as are the expression and distribution of the ECS receptors, biosynthetic and degradative enzymes.
      • Marquéz L.
      • Suárez J.
      • Iglesias M.
      • Bermudez-Silva F.J.
      • de Fonseca F.R.
      • Andreu M.
      Ulcerative colitis induces changes on the expression of the endocannabinoid system in the human colonic tissue.
      Although it is tempting to speculate that these changes contribute to the breakdown in homeostasis in these diseases, this remains to be directly demonstrated.
      Szabady et al
      • Szabady R.L.
      • Louissaint C.
      • Lubben A.
      • Xie B.
      • Reeksting S.
      • Tuohy C.
      • Demma Z.
      • Foley S.E.
      • Faherty C.S.
      • Llanos-Chea A.
      • Olive A.J.
      • Mrsny R.J.
      • McCormick B.A.
      Intestinal P-glycoprotein exports endocannabinoids to prevent inflammation and maintain homeostasis.
      did not find a role for the NAE, PEA, in inhibiting neutrophil migration; however, PEA has been shown by others to be a potent anti-inflammatory mediator acting via CB2 receptors and, in addition, GPR55 and PPARα, as well as through the modulation of TRPV1.
      • Borrelli F.
      • Romano B.
      • Petrosino S.
      • Pagano E.
      • Capasso R.
      • Coppola D.
      • Battista G.
      • Orlando P.
      • Di Marzo V.
      • Izzo A.A.
      Palmitoylethanolamide, a naturally occurring lipid, is an orally effective intestinal anti-inflammatory agent.
      In elegant work, Esposito et al
      • Esposito G.
      • Capoccia E.
      • Turco F.
      • Palumbo I.
      • Lu J.
      • Steardo A.
      • Cuomo R.
      • Sarnelli G.
      • Steardo L.
      Palmitoylethanolamide improves colon inflammation through an enteric glia/toll like receptor 4-dependent PPAR-α activation.
      demonstrated that PEA dose-dependently reduced colonic damage, inflammatory mediator expression and release, and immune cell infiltration in DSS colitis and inflammatory mediator expression and release in biopsy samples from patients with ulcerative colitis via PPARα activation. They demonstrated that the effects of PEA were mediated through an action on enteric glia by reducing the expression of toll-like receptor 4 and S100B.
      • Esposito G.
      • Capoccia E.
      • Turco F.
      • Palumbo I.
      • Lu J.
      • Steardo A.
      • Cuomo R.
      • Sarnelli G.
      • Steardo L.
      Palmitoylethanolamide improves colon inflammation through an enteric glia/toll like receptor 4-dependent PPAR-α activation.
      In an interesting extension of this work, this group recently developed a probiotic-based delivery system for PEA. Using genetic engineering, they developed a strain of Lactobacillus paracasei with the human NAPE-PLD gene inserted into it to produce an in situ delivery system for the release of PEA in the GI tract. They demonstrated that this approach was effective in a mouse model of Clostridium difficile colitis where colonic damage, inflammatory mediator release, and tight junction protein expression were all improved.
      • Esposito G.
      • Corpetti C.
      • Pesce M.
      • Seguella L.
      • Annunziata G.
      • Del Re A.
      • Vincenzi M.
      • Lattanzi R.
      • Lu J.
      • Sanseverino W.
      • Sarnelli G.
      A palmitoylethanolamide producing lactobacillus paracasei improves clostridium difficile toxin A-induced colitis.
      It was also similarly effective in DSS colitis.
      • Esposito G.
      • Corpetti C.
      • Pesce M.
      • Seguella L.
      • Annunziata G.
      • Del Re A.
      • Vincenzi M.
      • Lattanzi R.
      • Lu J.
      • Sanseverino W.
      • Sarnelli G.
      A palmitoylethanolamide producing lactobacillus paracasei improves clostridium difficile toxin A-induced colitis.
      The effects of the probiotic bacterium were abolished in PPARα knockout mice, suggesting they were mediated by PEA. However, because NAPE-PLD can synthesize a variety of NAEs and these were not assessed, it remains to be determined whether the effects observed are solely mediated by PEA. These studies placed enteric glia as critically important cellular intermediaries in the regulation of intestinal inflammation and homeostasis, a role that is gaining increasing recognition and clinical relevance.
      • Progatzky F.
      • Shapiro M.
      • Chng S.H.
      • Garcia-Cassani B.
      • Classon C.H.
      • Sevgi S.
      • Laddach A.
      • Bon-Frauches A.C.
      • Lasrado R.
      • Rahim M.
      • Amaniti E.M.
      • Boeing S.
      • Shah K.
      • Entwistle L.J.
      • Suárez-Bonnet A.
      • Wilson M.S.
      • Stockinger B.
      • Pachnis V.
      Regulation of intestinal immunity and tissue repair by enteric glia.
      • Benvenuti L.
      • D’antongiovanni V.
      • Pellegrini C.
      • Antonioli L.
      • Bernardini N.
      • Blandizzi C.
      • Fornai M.
      Enteric glia at the crossroads between intestinal immune system and epithelial barrier: implications for parkinson disease.
      • Seguella L.
      • Gulbransen B.D.
      Enteric glial biology, intercellular signalling and roles in gastrointestinal disease.
      For example, it was recently shown that toll-like receptor 4 on enteric glia is critical for the development of necrotizing enterocolitis.
      • Kovler M.L.
      • Gonzalez Salazar A.J.
      • Fulton W.B.
      • Lu P.
      • Yamaguchi Y.
      • Zhou Q.
      • Sampah M.
      • Ishiyama A.
      • Prindle T.
      • Wang S.
      • Jia H.
      • Wipf P.
      • Sodhi C.P.
      • Hackam D.J.
      Toll-like receptor 4–mediated enteric glia loss is critical for the development of necrotizing enterocolitis.
      Little is known about how the ECS regulates enteric glial function. Sharkey and colleagues showed that CB2 receptors could attenuate activation of enteric glia, which is consistent with other actions of cannabinoids, but beyond that this remains an area for further investigation.
      • Duncan M.
      • Mouihate A.
      • Mackie K.
      • Keenan C.M.
      • Buckley N.E.
      • Davison J.S.
      • Patel K.D.
      • Pittman Q.J.
      • Sharkey K.A.
      Cannabinoid CB 2 receptors in the enteric nervous system modulate gastrointestinal contractility in lipopolysaccharide-treated rats.
      The anti-inflammatory actions of PEA in the gut have been extended to intestinal inflammation in mouse models of Alzheimer’s disease, radiation injury, and ischemia-reperfusion injury.
      • D’Antongiovanni V.
      • Pellegrini C.
      • Antonioli L.
      • Benvenuti L.
      • Di Salvo C.
      • Flori L.
      • Piccarducci R.
      • Daniele S.
      • Martelli A.
      • Calderone V.
      • Martini C.
      • Fornai M.
      Palmitoylethanolamide counteracts enteric inflammation and bowel motor dysfunctions in a mouse model of Alzheimer’s disease.
      • Wang J.
      • Zheng J.
      • Kulkarni A.
      • Wang W.
      • Garg S.
      • Prather P.L.
      • Hauer-Jensen M.
      Palmitoylethanolamide regulates development of intestinal radiation injury in a mast cell-dependent manner.
      • Di Paola R.
      • Impellizzeri D.
      • Torre A.
      • Mazzon E.
      • Cappellani A.
      • Faggio C.
      • Esposito E.
      • Trischitta F.
      • Cuzzocrea S.
      Effects of palmitoylethanolamide on intestinal injury and inflammation caused by ischemia-reperfusion in mice.
      Although the actions of PEA are terminated by hydrolysis by FAAH, they are also regulated by N-acylethanolamine-hydrolyzing acid amidase (NAAA).
      • Alhouayek M.
      • Bottemanne P.
      • Subramanian K.V.
      • Lambert D.M.
      • Makriyannis A.
      • Cani P.D.
      • Muccioli G.G.
      N-acylethanolamine-hydrolyzing acid amidase inhibition increases colon N-palmitoylethanolamine levels and counteracts murine colitis.
      Inhibiting NAAA elevates levels of PEA, while not altering those of AEA. In TNBS colitis in mice, a NAAA inhibitor significantly reduced the degree of colitis and the release of inflammatory mediators,
      • Alhouayek M.
      • Bottemanne P.
      • Subramanian K.V.
      • Lambert D.M.
      • Makriyannis A.
      • Cani P.D.
      • Muccioli G.G.
      N-acylethanolamine-hydrolyzing acid amidase inhibition increases colon N-palmitoylethanolamine levels and counteracts murine colitis.
      indicating that endogenously produced PEA can regulate inflammation and mucosal integrity. Interestingly, the related NAE, OEA, is also anti-inflammatory in DSS colitis,
      • Lama A.
      • Provensi G.
      • Amoriello R.
      • Pirozzi C.
      • Rani B.
      • Mollica M.P.
      • Raso G.M.
      • Ballerini C.
      • Meli R.
      • Passani M.B.
      The anti-inflammatory and immune-modulatory effects of OEA limit DSS-induced colitis in mice.
      although the receptor mechanisms remain to be determined.
      Activation of CB1 receptors in the gut is also anti-inflammatory in models of experimental colitis,
      • D’Argenio G.
      • Valenti M.
      • Scaglione G.
      • Cosenza V.
      • Sorrentini I.
      • Di Marzo V.
      Up-regulation of anandamide levels as an endogenous mechanism and a pharmacological strategy to limit colon inflammation.
      ,
      • Storr M.A.
      • Keenan C.M.
      • Emmerdinger D.
      • Zhang H.
      • Yüce B.
      • Sibaev A.
      • Massa F.
      • Buckley N.E.
      • Lutz B.
      • Göke B.
      • Brand S.
      • Patel K.D.
      • Sharkey K.A.
      Targeting endocannabinoid degradation protects against experimental colitis in mice: involvement of CB1 and CB2 receptors.
      ,
      • Massa F.
      • Marsicano G.
      • Hermann H.
      • Cannich A.
      • Monory K.
      • Cravatt B.F.
      • Ferri G.-L.
      • Sibaev A.
      • Storr M.
      • Lutz B.
      The endogenous cannabinoid system protects against colonic inflammation.
      and consistent with those observations, CB1 receptor knockout mice have exacerbated disease.
      • Engel M.A.
      • Kellerman C.A.
      • Burnat G.
      • Hahn E.G.
      • Rau T.
      • Konturek P.C.
      Mice lacking cannabinoid CB1-, CB2-receptors or both receptors show increased susceptibility to trinitrobenzene sulfonic acid (TNBS)-induced colitis.
      ,
      • Massa F.
      • Marsicano G.
      • Hermann H.
      • Cannich A.
      • Monory K.
      • Cravatt B.F.
      • Ferri G.-L.
      • Sibaev A.
      • Storr M.
      • Lutz B.
      The endogenous cannabinoid system protects against colonic inflammation.
      Although CB1 receptors are expressed throughout the GI tract,
      • Sharkey K.A.
      • Wiley J.W.
      The role of the endocannabinoid system in the brain–gut axis.
      ,
      • Trautmann S.M.
      • Sharkey K.A.
      The endocannabinoid system and its role in regulating the intrinsic neural circuitry of the gastrointestinal tract.
      a peripherally restricted CB1 receptor agonist does not protect against colitis,
      • Fichna J.
      • Bawa M.
      • Thakur G.A.
      • Tichkule R.
      • Makriyannis A.
      • McCafferty D.M.
      • Sharkey K.A.
      • Storr M.
      Cannabinoids alleviate experimentally induced intestinal inflammation by acting at central and peripheral receptors.
      ,
      • Cluny N.L.
      • Keenan C.M.
      • Duncan M.
      • Fox A.
      • Lutz B.
      • Sharkey K.A.
      Naphthalen-1-yl-(4-pentyloxynaphthalen-1-yl)methanone (SAB378), a peripherally restricted cannabinoid CB1/CB2 receptor agonist, inhibits gastrointestinal motility but has no effect on experimental colitis in mice.
      whereas a centrally administered CB1 agonist was protective.
      • Fichna J.
      • Bawa M.
      • Thakur G.A.
      • Tichkule R.
      • Makriyannis A.
      • McCafferty D.M.
      • Sharkey K.A.
      • Storr M.
      Cannabinoids alleviate experimentally induced intestinal inflammation by acting at central and peripheral receptors.
      This puzzling finding requires further study.
      Endogenous 2-AG levels can be selectively elevated by inhibiting the primary enzyme responsible for its metabolism, MAGL. Elevating levels of 2-AG using a pharmacologic MAGL inhibitor attenuated murine TNBS colitis and significantly improved intestinal barrier function.
      • Alhouayek M.
      • Lambert D.M.
      • Delzenne N.M.
      • Cani P.D.
      • Muccioli G.G.
      Increasing endogenous 2-arachidonoylglycerol levels counteracts colitis and related systemic inflammation.
      Interestingly, the effects of the MAGL inhibitor were blocked by both CB1 and CB2 receptor antagonists, as they are when FAAH inhibitors are used to elevate endogenous endocannabinoids.
      • Storr M.A.
      • Keenan C.M.
      • Emmerdinger D.
      • Zhang H.
      • Yüce B.
      • Sibaev A.
      • Massa F.
      • Buckley N.E.
      • Lutz B.
      • Göke B.
      • Brand S.
      • Patel K.D.
      • Sharkey K.A.
      Targeting endocannabinoid degradation protects against experimental colitis in mice: involvement of CB1 and CB2 receptors.
      Although it seems likely that some of the effects of inhibiting MAGL are localized to the gut, it may be that the CB1 effects are also (or only) centrally mediated. This remains to be determined and is an exciting area for future study.
      Mice with a genetic deletion of MAGL (Mgll knockout mice) have chronic elevations of 2-AG, leading to the desensitization and accumulation of CB1 receptors and functional inhibition of CB1 receptors in the gut and brain.
      • Taschler U.
      • Eichmann T.O.
      • Radner F.P.W.
      • Grabner G.F.
      • Wolinski H.
      • Storr M.
      • Lass A.
      • Schicho R.
      • Zimmermann R.
      Monoglyceride lipase deficiency causes desensitization of intestinal cannabinoid receptor type 1 and increased colonic μ-opioid receptor sensitivity.
      ,
      • Schlosburg J.E.
      • Blankman J.L.
      • Long J.Z.
      • Nomura D.K.
      • Pan B.
      • Kinsey S.G.
      • Nguyen P.T.
      • Ramesh D.
      • Booker L.
      • Burston J.J.
      • Thomas E.A.
      • Selley D.E.
      • Sim-Selley L.J.
      • Liu Q.S.
      • Lichtman A.H.
      • Cravatt B.F.
      Chronic monoacylglycerol lipase blockade causes functional antagonism of the endocannabinoid system.
      Using this model, Ellerman et al
      • Ellermann M.
      • Pacheco A.R.
      • Jimenez A.G.
      • Russell R.M.
      • Cuesta S.
      • Kumar A.
      • Zhu W.
      • Vale G.
      • Martin S.A.
      • Raj P.
      • McDonald J.G.
      • Winter S.E.
      • Sperandio V.
      Endocannabinoids inhibit the induction of virulence in enteric pathogens.
      recently showed that these mice were protected from the effects of enteric bacterial infection with the attaching and effacing enteric pathogen Citrobacter rodentium, which causes a breakdown of barrier function and marked intestinal inflammation. They made the remarkable discovery that the effect was mediated via an action of 2-AG on the virulence programs essential for successful bacterial infection.
      • Ellermann M.
      • Pacheco A.R.
      • Jimenez A.G.
      • Russell R.M.
      • Cuesta S.
      • Kumar A.
      • Zhu W.
      • Vale G.
      • Martin S.A.
      • Raj P.
      • McDonald J.G.
      • Winter S.E.
      • Sperandio V.
      Endocannabinoids inhibit the induction of virulence in enteric pathogens.
      2-AG works by antagonizing QseC, the bacterial histidine kinase that promotes the activation of the type III secretion system, used to infect host cells. QseC is a quorum sensing receptor that was also discovered to be a bacterial adrenergic receptor, responding to epinephrine and norepinephrine, to enhance bacterial virulence.
      • Moreira C.G.
      • Russell R.
      • Mishra A.A.
      • Narayanan S.
      • Ritchie J.M.
      • Waldor M.K.
      • Curtis M.M.
      • Winter S.E.
      • Weinshenker D.
      • Sperandio V.
      Bacterial adrenergic sensors regulate virulence of enteric pathogens in the gut.
      ,
      • Clarke M.B.
      • Hughes D.T.
      • Zhu C.
      • Boedeker E.C.
      • Sperandio V.
      The QseC sensor kinase: a bacterial adrenergic receptor.
      It therefore appears that 2-AG counteracts the effects of adrenergic stimuli, reducing virulence of C rodentium, thereby also providing an interesting example of interkingdom signaling in which host signaling molecules have the capacity to regulate the degree of bacterial virulence resulting in the maintenance of intestinal homeostasis.
      Taken together, these data strongly suggest that the local production and release of endocannabinoids in the GI tract maintain an anti-inflammatory environment in the face of enteric infection or mucosal aggravation and damage. Future studies aimed at discovering the cellular sources of endocannabinoids and the mechanisms that govern their production and release in the intestines are critical for a full understanding of the role of the ECS in regulating intestinal homeostasis in response to bacterial pathogens and intestinal inflammation.

      Reciprocal Regulation of the Endocannabinoid System and the Gut Microbiome

      The gut microbiota is a critical environmental determinant of host physiology. Studies using germ-free mice, or mice treated with antibiotics to deplete the gut microbiota, have revealed that the microbiota shapes gut function and enteric neural control mechanisms.
      • Vicentini F.A.
      • Keenan C.M.
      • Wallace L.E.
      • Woods C.
      • Cavin J.B.
      • Flockton A.R.
      • Macklin W.B.
      • Belkind-Gerson J.
      • Hirota S.A.
      • Sharkey K.A.
      Intestinal microbiota shapes gut physiology and regulates enteric neurons and glia.
      • Muller P.A.
      • Matheis F.
      • Schneeberger M.
      • Kerner Z.
      • Jové V.
      • Mucida D.
      Microbiota-modulated CART+ enteric neurons autonomously regulate blood glucose.
      • Obata Y.
      • Pachnis V.
      The effect of microbiota and the immune system on the development and organization of the enteric nervous system.
      • Obata Y.
      • Castaño Á.
      • Boeing S.
      • Bon-Frauches A.C.
      • Fung C.
      • Fallesen T.
      • de Agüero M.G.
      • Yilmaz B.
      • Lopes R.
      • Huseynova A.
      • Horswell S.
      • Maradana M.R.
      • Boesmans W.
      • Vanden Berghe P.
      • Murray A.J.
      • Stockinger B.
      • Macpherson A.J.
      • Pachnis V.
      Neuronal programming by microbiota regulates intestinal physiology.
      • Niesler B.
      • Kuerten S.
      • Demir I.E.
      • Schäfer K.H.
      Disorders of the enteric nervous system: a holistic view.
      • De Vadder F.
      • Grasset E.
      • Holm L.M.
      • Karsenty G.
      • Macpherson A.J.
      • Olofsson L.E.
      • Bäckhed F.
      Gut microbiota regulates maturation of the adult enteric nervous system via enteric serotonin networks.
      Microbial dysbiosis is associated with a breakdown in epithelial barrier function and is associated with both local GI diseases, eg, IBD, and various systemic conditions, eg, obesity and diabetes. Investigations into the regulation of the ECS by the gut microbiome and vice versa are at an early stage, but some intriguing results have emerged that support the general hypothesis that the ECS regulates intestinal homeostasis through interactions with the microbiota.
      In the absence of a gut microbiota, significant changes in the expression of the genes for receptors, biosynthetic and degradative enzymes of the ECS were observed throughout the gut.
      • Manca C.
      • Boubertakh B.
      • Leblanc N.
      • Deschênes T.
      • Lacroix S.
      • Martin C.
      • Houde A.
      • Veilleux A.
      • Flamand N.
      • Muccioli G.G.
      • Raymond F.
      • Cani P.D.
      • Di Marzo V.
      • Silvestri C.
      Germ-free mice exhibit profound gut microbiota-dependent alterations of intestinal endocannabinoidome signaling.
      For example, cnr1 (CB1 receptor) is markedly elevated in the colon of germ-free mice. Overall, more striking changes were seen in the small intestine compared with the colon, and when mice were younger (4 weeks compared with 13 weeks of age). In concert with these changes, there were also alterations in the levels of endocannabinoids, NAEs, and other related lipid mediators, to a variable degree along the length of the gut.
      • Manca C.
      • Boubertakh B.
      • Leblanc N.
      • Deschênes T.
      • Lacroix S.
      • Martin C.
      • Houde A.
      • Veilleux A.
      • Flamand N.
      • Muccioli G.G.
      • Raymond F.
      • Cani P.D.
      • Di Marzo V.
      • Silvestri C.
      Germ-free mice exhibit profound gut microbiota-dependent alterations of intestinal endocannabinoidome signaling.
      Most of the changes observed were reversible when a normal gut flora was introduced using fecal microbial transplant.
      • Manca C.
      • Boubertakh B.
      • Leblanc N.
      • Deschênes T.
      • Lacroix S.
      • Martin C.
      • Houde A.
      • Veilleux A.
      • Flamand N.
      • Muccioli G.G.
      • Raymond F.
      • Cani P.D.
      • Di Marzo V.
      • Silvestri C.
      Germ-free mice exhibit profound gut microbiota-dependent alterations of intestinal endocannabinoidome signaling.
      However, it should be noted that the germ-free animals were only recolonized in these studies for 1 week, which may not have been sufficient for stable microbial recolonization. Nevertheless, these data demonstrate that the gut microbiota is directly impacting all aspects of the ECS.
      The ECS has also been examined after antibiotic administration was used to deplete the gut microbiota.
      • Guida F.
      • Turco F.
      • Iannotta M.
      • De Gregorio D.
      • Palumbo I.
      • Sarnelli G.
      • Furiano A.
      • Napolitano F.
      • Boccella S.
      • Luongo L.
      • Mazzitelli M.
      • Usiello A.
      • De Filippis F.
      • Iannotti F.A.
      • Piscitelli F.
      • Ercolini D.
      • de Novellis V.
      • Di Marzo V.
      • Cuomo R.
      • Maione S.
      Antibiotic-induced microbiota perturbation causes gut endocannabinoidome changes, hippocampal neuroglial reorganization and depression in mice.
      • Aguilera M.
      • Vergara P.
      • Martínez V.
      Stress and antibiotics alter luminal and wall-adhered microbiota and enhance the local expression of visceral sensory-related systems in mice.
      • Aguilera M.
      • Cerdà-Cuéllar M.
      • Martínez V.
      Antibiotic-induced dysbiosis alters host-bacterial interactions and leads to colonic sensory and motor changes in mice.
      Modest changes to AEA levels were noted along the length of small intestine with no changes to 2-AG
      • Guida F.
      • Turco F.
      • Iannotta M.
      • De Gregorio D.
      • Palumbo I.
      • Sarnelli G.
      • Furiano A.
      • Napolitano F.
      • Boccella S.
      • Luongo L.
      • Mazzitelli M.
      • Usiello A.
      • De Filippis F.
      • Iannotti F.A.
      • Piscitelli F.
      • Ercolini D.
      • de Novellis V.
      • Di Marzo V.
      • Cuomo R.
      • Maione S.
      Antibiotic-induced microbiota perturbation causes gut endocannabinoidome changes, hippocampal neuroglial reorganization and depression in mice.
      ; however, there were marked changes to NAEs and other endocannabinoidome ligands. Interestingly, increased antibiotic treatment resulted in increased CB2 receptor expression.
      • Aguilera M.
      • Vergara P.
      • Martínez V.
      Stress and antibiotics alter luminal and wall-adhered microbiota and enhance the local expression of visceral sensory-related systems in mice.
      ,
      • Aguilera M.
      • Cerdà-Cuéllar M.
      • Martínez V.
      Antibiotic-induced dysbiosis alters host-bacterial interactions and leads to colonic sensory and motor changes in mice.
      CB2 receptor expression was further increased in animals exposed to antibiotics who were also exposed to a period of water-avoidance stress,
      • Aguilera M.
      • Vergara P.
      • Martínez V.
      Stress and antibiotics alter luminal and wall-adhered microbiota and enhance the local expression of visceral sensory-related systems in mice.
      which is illustrative of how the ECS responds to perturbations that lead to elevated visceral sensitivity.
      Taken together, these studies reveal how the ECS is relatively altered by marked changes in the microbial environment, although the mechanism underlying these alterations remains to be determined. Importantly, microbial changes to endocannabinoid signaling result in biologically relevant functional effects at the level of the gut and the brain; it should be noted that commensal bacteria make endocannabinoid ligands,
      • Cohen L.J.
      • Esterhazy D.
      • Kim S.H.
      • Lemetre C.
      • Aguilar R.R.
      • Gordon E.A.
      • Pickard A.J.
      • Cross J.R.
      • Emiliano A.B.
      • Han S.M.
      • Chu J.
      • Vila-Farres X.
      • Kaplitt J.
      • Rogoz A.
      • Calle P.Y.
      • Hunter C.
      • Bitok J.K.
      • Brady S.F.
      Commensal bacteria make GPCR ligands that mimic human signalling molecules.
      although their role in GI physiology remains obscure.
      Administration of prebiotics or probiotics alters the composition of the gut microbiota (transiently) and can be used therapeutically. The benefits of probiotics may be mediated, at least in part, by the ECS. For example, treatment with the probiotic Lactobacillus acidophilus up-regulated CB2 receptors on intestinal epithelial cells. When animals with visceral hypersensitivity were treated with this probiotic, it gave rise to a pronounced visceral analgesia to colorectal distention, sensitive to CB2 receptor antagonism, assessed in vivo.
      • Rousseaux C.
      • Thuru X.
      • Gelot A.
      • Barnich N.
      • Neut C.
      • Dubuquoy L.
      • Dubuquoy C.
      • Merour E.
      • Geboes K.
      • Chamaillard M.
      • Ouwehand A.
      • Leyer G.
      • Carcano D.
      • Colombel J.F.
      • Ardid D.
      • Desreumaux P.
      Lactobacillus acidophilus modulates intestinal pain and induces opioid and cannabinoid receptors.
      The exact mechanisms behind these interesting effects remain to be determined because the source and nature of the ligand activating the CB2 receptors were not determined. However, the inhibitory actions of CB2 receptor activation are consistent with recent studies that show that optogenetic inhibition of the colonic epithelium reduces visceral hypersensitivity in mice with DSS colitis.
      • Najjar S.A.
      • Ejoh L.L.
      • Loeza-Alcocer E.
      • Edwards B.S.
      • Smith-Edwards K.M.
      • Epouhe A.Y.
      • Gold M.S.
      • Davis B.M.
      • Albers K.M.
      Optogenetic inhibition of the colon epithelium reduces hypersensitivity in a mouse model of inflammatory bowel disease.
      In another example, treatment with the probiotic Lactobacillus plantarumWJL reduced despair behavior and increased hippocampal neurogenesis associated with a chronic stress paradigm by altering endocannabinoid levels in the hippocampus.
      • Chevalier G.
      • Siopi E.
      • Guenin-Macé L.
      • Pascal M.
      • Laval T.
      • Rifflet A.
      • Boneca I.G.
      • Demangel C.
      • Colsch B.
      • Pruvost A.
      • Chu-Van E.
      • Messager A.
      • Leulier F.
      • Lepousez G.
      • Eberl G.
      • Lledo P.M.
      Effect of gut microbiota on depressive-like behaviors in mice is mediated by the endocannabinoid system.
      The gut-brain pathways involved in these effects remain to be determined.
      Just as probiotics can alter the ECS in ways that are beneficial, altering the microbiota can also give rise to effects that are detrimental. Recently, Markey et al
      • Markey L.
      • Hooper A.
      • Melon L.C.
      • Baglot S.
      • Hill M.N.
      • Maguire J.
      • Kumamoto C.A.
      Colonization with the commensal fungus Candida albicans perturbs the gut-brain axis through dysregulation of endocannabinoid signaling.
      colonized healthy mice with the commensal fungus Candida albicans for 48 hours. Candida colonization caused no changes to the cecal bacterial populations in the gut and no intestinal inflammation. However, there were marked increases in anxiety-like behavior, accompanied by elevations in plasma corticosterone that were inversely correlated with forebrain AEA levels. Colonization with Candida disrupted the metabolism of endocannabinoids in the gut, notably the NAEs. Consistent with these observations, when mice were treated with the FAAH inhibitor URB597, corticosterone levels were reduced to control levels, as was anxiety-like behavior. This finding is illustrative of the importance of the microbiota-gut-brain axis in regulating behavior and how the ECS is an important component of this signaling system.
      In an interesting study in human subjects, Vijay et al
      • Vijay A.
      • Kouraki A.
      • Gohir S.
      • Turnbull J.
      • Kelly A.
      • Chapman V.
      • Barrett D.A.
      • Bulsiewicz W.J.
      • Valdes A.M.
      The anti-inflammatory effect of bacterial short chain fatty acids is partially mediated by endocannabinoids.
      studied the relationships between the gut microbiome, ECS, and inflammatory cytokines after 6-week exercise intervention. They demonstrated that under baseline conditions, the NAEs were positively associated with bacterial alpha diversity and with short-chain fatty acid producing bacterial species including Bifidobacterium and Faecalibacterium and negatively associated with the pathogenic bacterium Escherichia Shigella. The NAEs increased significantly after the exercise intervention. Changes in AEA correlated with elevated butyrate levels, increases in AEA and PEA correlated with reductions in the inflammatory cytokines, tumor necrosis factor and interleukin 6, whereas 2-AG and OEA levels were correlated with anti-inflammatory cytokine interleukin 10.
      • Vijay A.
      • Kouraki A.
      • Gohir S.
      • Turnbull J.
      • Kelly A.
      • Chapman V.
      • Barrett D.A.
      • Bulsiewicz W.J.
      • Valdes A.M.
      The anti-inflammatory effect of bacterial short chain fatty acids is partially mediated by endocannabinoids.
      This study again highlights the interactions between the ECS and the gut microbiota and reveals that the ECS is involved in mediating homeostatic anti-inflammatory actions in humans.
      The connection between gut microbiota and the ECS has been studied in the context of the regulation of energy homeostasis. Diet-induced obesity in mice is associated with an altered gut microbiota. Chronic administration of the CB1/CB2 partial agonist, THC, prevented the change in gut microbiota in the DIO mice.
      • Cluny N.L.
      • Keenan C.M.
      • Reimer R.A.
      • Foll B Le
      • Sharkey K.A.
      Prevention of diet-induced obesity effects on body weight and gut microbiota in mice treated chronically with Δ9-tetrahydrocannabinol.
      Chronic THC administration in DIO mice increases the abundance of Akkermansia muciniphila.
      • Cluny N.L.
      • Keenan C.M.
      • Reimer R.A.
      • Foll B Le
      • Sharkey K.A.
      Prevention of diet-induced obesity effects on body weight and gut microbiota in mice treated chronically with Δ9-tetrahydrocannabinol.
      Interestingly, THC had no effect on gut microbiota in lean mice.
      • Cluny N.L.
      • Keenan C.M.
      • Reimer R.A.
      • Foll B Le
      • Sharkey K.A.
      Prevention of diet-induced obesity effects on body weight and gut microbiota in mice treated chronically with Δ9-tetrahydrocannabinol.
      Administration of A muciniphila in DIO mice also increases levels of 2-AG, 2-OG, and 2-PG in the gut.
      • Everard A.
      • Belzer C.
      • Geurts L.
      • Ouwerkerk J.P.
      • Druart C.
      • Bindels L.B.
      • Guiot Y.
      • Derrien M.
      • Muccioli G.G.
      • Delzenne N.M.
      • De Vos W.M.
      • Cani P.D.
      Cross-talk between Akkermansia muciniphila and intestinal epithelium controls diet-induced obesity.
      Furthermore, blockade of the CB1 receptor with the CB1 antagonist rimonabant increased the abundance of A muciniphila in the gut but also led to a decreased relative abundance of Lachnospiraceae and Erysipelotrichaceae.
      • Mehrpouya-Bahrami P.
      • Chitrala K.N.
      • Ganewatta M.S.
      • Tang C.
      • Murphy E.A.
      • Enos R.T.
      • Velazquez K.T.
      • McCellan J.
      • Nagarkatti M.
      • Nagarkatti P.
      Blockade of CB1 cannabinoid receptor alters gut microbiota and attenuates inflammation and diet-induced obesity.
      Insights into how the ECS might regulate the gut microbiota in the context of a high-fat diet were obtained by Everard et al
      • Everard A.
      • Plovier H.
      • Rastelli M.
      • Van Hul M.
      • de Wouters d’Oplinter A.
      • Geurts L.
      • Druart C.
      • Robine S.
      • Delzenne N.M.
      • Muccioli G.G.
      • de Vos W.M.
      • Luquet S.
      • Flamand N.
      • Di Marzo V.
      • Cani P.D.
      Intestinal epithelial N-acylphosphatidylethanolamine phospholipase D links dietary fat to metabolic adaptations in obesity and steatosis.
      using molecular genetics. They selectively knocked out nape-pld (the gene for NAPE-PLD) in intestinal epithelial cells in mice fed a high-fat diet. This resulted in marked changes to the gut microbiota composition that were shown not to be due to the diet per se. These data suggest that epithelially derived NAEs are the mediators of signaling that ultimately alter microbial composition. It remains to be shown how this occurs, but one possibility is through the regulation of local innate immune mechanisms.
      Specific changes in the gut microbiota through several models (eg, prebiotic treatment, high-fat diet, antibiotic treatment, and germ-free mice) selectively alter CB1, FAAH, and MAGL mRNA expression in the colon.
      • Muccioli G.G.
      • Naslain D.
      • Bäckhed F.
      • Reigstad C.S.
      • Lambert D.M.
      • Delzenne N.M.
      • Cani P.D.
      The endocannabinoid system links gut microbiota to adipogenesis.
      The gut microbiota appears to modulate intestinal endocannabinoid tone in the colon but has no effect in the small intestine, which is likely due to the greater bacterial load in the colon. Although evidence suggests that changes in the composition of the gut microbiota affect colonic endocannabinoid tone, the exact mechanism involved in this regulation remains unknown.

      Conclusions and Perspective

      A summary of the material we have presented in this review is presented in Figure 4. Although there are numerous unanswered questions, the wealth of evidence accumulated to date suggests that the ECS is intimately involved in the physiological processes that underlie the control of intestinal homeostasis. The pivotal role the ECS plays in the maintenance of gut barrier integrity is complex and multifaceted because it responds to internal (microbial) and external (diet, stress, etc) environmental factors, while also serving as a homeostatic effector system. A lot of the evidence for the role of the ECS is based on the use of pharmacologic tools or a global knockout of the CB receptors or degradative enzymes. The application of cell-specific gene deletion technologies is required to causally determine the (patho)physiological role of the ECS. Where this has been used, for example to understand the role of epithelial NAPE-PLD in DIO,
      • Everard A.
      • Plovier H.
      • Rastelli M.
      • Van Hul M.
      • de Wouters d’Oplinter A.
      • Geurts L.
      • Druart C.
      • Robine S.
      • Delzenne N.M.
      • Muccioli G.G.
      • de Vos W.M.
      • Luquet S.
      • Flamand N.
      • Di Marzo V.
      • Cani P.D.
      Intestinal epithelial N-acylphosphatidylethanolamine phospholipase D links dietary fat to metabolic adaptations in obesity and steatosis.
      very novel findings have emerged that greatly advanced our understanding of the physiology of the ECS.
      Figure thumbnail gr4
      Figure 4Overview of the endocannabinoid system in the gut. Endocannabinoids in the intestine are produced primarily by enteric neurons and by subpopulations of extrinsic neurons. Some epithelial subtypes, including goblet and Paneth cells, may also produce endocannabinoids, based on the presence of synthetic enzymes identified in single cell expression studies. The main endocannabinoids are anandamide (AEA) and 2-arachidonoylglycerol (2-AG). Other N-acylethanolamides and acylglycerols are endocannabinoids that act to regulate gut functions. Endocannabinoids act at CB1 and CB2 receptors to regulate various functions in the gut. Various immune cells express CB2 receptors, which function primarily to dampen immune responses. Enteric neurons express CB1 receptors, which modulate enteric neural control of various gastrointestinal functions. Various epithelial subtypes express CB1 receptor under physiological conditions, which acts to suppress secretory responses. CB1 activation also decreases epithelial permeability by altering tight junction protein expression and localization. Endocannabinoids have a reciprocal relationship with the gut microbiota; endocannabinoids can affect the composition of the microbiota, and different commensals (bacteria, fungi) can alter the endocannabinoid system. 2-OG, 2-oleoylglycerol; 2-PG, 2-palmitoylglycerol; LEA, N-linoleoylethanolamide; OEA, N-oleoylethanolamide; PEA, N-palmitoylenthanolamide; SEA, N-stearoylethanolamide. Figure created with BioRender.com.
      An important consideration of the ECS that needs to be borne in mind is the context dependent nature of CB receptor activation by endocannabinoids. By this we refer to the fact that the 2 major endocannabinoids AEA and 2-AG are partial and full agonists of the CB1 receptor, respectively, and as we have discussed, they can have opposite effects in the GI tract or individual effects rather than identical actions. Because of the complexity of lipid signaling in inflammatory states in particular, where there may be a diversity of precursors according to the expression of the rate limiting enzymes for the synthesis of the lipid moieties, then ECS signaling may be altered in ways that are not easy to predict. Furthermore, many experimental, synthetic cannabinoids have biased signaling effects at CB receptors that do not precisely reflect signaling effects observed with endocannabinoids. Hence, future research that takes an integrative lipidomic approach to studies of the GI tract in health and disease are much needed to reconcile some of the disparate observations in the literature.
      The ECS has been proposed as a good therapeutic target for the treatment of GI inflammatory diseases and conditions that involve a breakdown in intestinal homeostasis.
      • Picardo S.
      • Kaplan G.G.
      • Sharkey K.A.
      • Seow C.H.
      Insights into the role of cannabis in the management of inflammatory bowel disease.
      • Kienzl M.
      • Storr M.
      • Schicho R.
      Cannabinoids and opioids in the treatment of inflammatory bowel diseases.
      • Gotfried J.
      • Naftali T.
      • Schey R.
      Role of cannabis and its derivatives in gastrointestinal and hepatic disease.
      • Maselli D.B.
      • Camilleri M.
      Pharmacology, clinical effects, and therapeutic potential of cannabinoids for gastrointestinal and liver diseases.
      However, because of the ubiquitous distribution of the ECS in the GI tract and its complex physiology, careful consideration needs to be given to the best molecular target. This is currently challenging because the precise sites of endocannabinoid production are not well-understood, and a detailed description of the distribution of the biosynthetic and degradative enzymes for the expanded ECS is also lacking. Future studies addressing these limitations will greatly advance our understanding of the potential of the ECS to be selectively targeted for the treatment of disease as well as shedding new light on the physiology of the ECS in the GI tract.

      CRediT Authorship Contributions

      Hailey Cuddihey (Conceptualization: Supporting; Writing – original draft: Equal; Writing – review & editing: Equal)
      Wallace K. MacNaughton (Conceptualization: Supporting; Resources: Equal; Writing – original draft: Equal; Writing – review & editing: Equal)
      Keith A. Sharkey, PhD (Conceptualization: Lead; Resources: Lead; Writing – original draft: Equal; Writing – review & editing: Equal)

      Supplementary Material

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