Abnormal cannabidiol attenuates experimental colitis in mice, promotes wound healing and inhibits neutrophil recruitment
© The Author(s). 2016
Received: 18 November 2015
Accepted: 15 June 2016
Published: 14 July 2016
Non-psychotropic atypical cannabinoids have therapeutic potential in a variety of inflammatory conditions including those of the gastrointestinal tract. Here we examined the effects of the atypical cannabinoid abnormal cannabidiol (Abn-CBD) on wound healing, inflammatory cell recruitment and colitis in mice.
Colitis was induced in CD1 mice by a single intrarectal administration of trinitrobenzene sulfonic acid (TNBS, 4 mg/100 μl in 30 % ethanol) and Abn-CBD and/or the antagonists O-1918 (Abd-CBD), AM251 (CB1 receptor) and AM630 (CB2 receptor), were administered intraperitoneally (all 5 mg/kg, twice daily for 3 days). The degree of colitis was assessed macro- and microscopically and tissue myeloperoxidase activity was determined. The effects of Abn-CBD on wound healing of endothelial and epithelial cells (LoVo) were assessed in a scratch injury assay. Human neutrophils were employed in Transwell assays or perfused over human umbilical vein endothelial cells (HUVEC) to study the effect of Abn-CBD on neutrophil accumulation and transmigration.
TNBS-induced colitis was attenuated by treatment with Abn-CBD. Histological, macroscopic colitis scores and tissue myeloperoxidase activity were significantly reduced. These effects were inhibited by O-1918, but not by AM630, and only in part by AM251. Wound healing of both HUVEC and LoVo cells was enhanced by Abn-CBD. Abn-CBD inhibited neutrophil migration towards IL-8, and dose-dependently inhibited accumulation of neutrophils on HUVEC.
Abn-CBD is protective against TNBS-induced colitis, promotes wound healing of endothelial and epithelial cells and inhibits neutrophil accumulation on HUVEC monolayers. Thus, the atypical cannabinoid Abn-CBD represents a novel potential therapeutic in the treatment of intestinal inflammatory diseases.
Cannabis has long been recognized in the Western world for its analgesic, appetite stimulant, antiemetic, muscle relaxant and anticonvulsant properties . Today it is known that plant-derived cannabinoids, or phytocannabinoids, such as Δ9-tetrahydrocannabinol (Δ9-THC) and cannabinol exert their therapeutic effects through the cannabinoid 1 (CB1) and CB2 receptors of the endocannabinoid system . Extracts of Cannabis have been used as therapeutics in gastrointestinal disorders, but the major pharmacologically active component, Δ9-THC has psychotropic properties which limit its suitability as a drug .
Aiming to circumvent the psychotropic side effects of cannabis, researchers have focused on the therapeutic potential of non-psychotropic cannabinoids, such as the phytocannabinoid cannabidiol (CBD). CBD has been shown to attenuate experimental colitis in mice, when administered topically or systemically . In addition, the anti-inflammatory and modest antioxidant properties of CBD make it a promising candidate for drug development to target a number of systemic diseases, including rheumatoid arthritis and atherosclerosis . Using CBD as the prototype, synthetic analogs have been developed such as the regio-isomer abnormal cannabidiol (Abn-CBD) and it’s close relative O-1602. These ‘atypical’ cannabinoids lack significant binding affinity to cannabinoid receptors, but act on novel targets such as the orphan receptor GPR55 [6, 7]. It has been demonstrated that CBD and O-1602 protect against experimentally induced colitis in mice, but their mechanisms of action requires further investigation, notably, as the protective properties of O-1602 are also observed in mice lacking the GPR55 gene [8, 9]. They might be conferred by GPR18, another target of O-1602 and the putative receptor of Abn-CBD . While studies have examined the vasodilatory and neuroprotective effects of Abn-CBD [7, 11–13], its potential role in the modulation of gastrointestinal inflammation has not been examined.
The aim of this study was to examine if the CBD analogue Abn-CBD has therapeutic potential in the treatment of gastrointestinal inflammation. We tested the hypothesis that Abn-CBD would reduce intestinal inflammation and accelerate epithelial would healing. We first examined the therapeutic effect of Abn-CBD in a murine model of experimentally induced colitis. The Abn-CBD receptor antagonist O-1918, and the CB1 and CB2 receptor antagonists, AM251 and AM630, respectively, were employed in order to elucidate, if Abn-CBD effects were CB1/CB2 dependent or conferred by other receptors. Next we examined the effects of the Abn-CBD on neutrophil recruitment, an important cellular mechanism of intestinal inflammation. Lastly, we studied the impact of Abn-CBD on endothelial and epithelial wound healing in vitro, to address a further potential therapeutic mechanism of action [14, 15].
Male CD1 mice (3 weeks old, weighing ~16 g) were purchased from Charles River (Saint-Constant, Quebec, Canada) and kept in-house for 2 weeks prior to experiments. Mice were housed in plastic sawdust floor cages at constant temperature (22 °C) and a 12:12-h light–dark cycle with access to standard laboratory chow and tap water ad libitum. Experimental procedures were approved by the University of Calgary Animal Care Committee and conducted according to guidelines of the Canadian Council on Animal Care.
Drugs and Pharmacological Treatments
Trinitrobenzene sulfonic acid (TNBS) was purchased from Sigma-Aldrich (Oakville, Ontario, Canada). Abn-CBD, dissolved in methyl acetate, O-1918 (1,3-Dimethoxy-5-methyl-2-[(1R,6R)-3-methyl-6-(1-methylethenyl)-2-cyclohexen-1-yl]benz-ene), AM630 (6-iodo-2-methyl-1-[2-(4-morpholinyl)ethyl-1H-indol-3-yl](4-methoxyphenyl)methanone) and AM251 (N-(Piperidin-1-yl)-5-(4-iodophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide) were obtained from Tocris Bioscience (Bristol, UK). Because of its toxicity, methyl acetate was evaporated prior to the in vivo experiments and ethanol was used instead as a solvent. Abn-CBD was then further diluted in Tween 80 (10 %) and sterile saline. Vehicle consisted of ethanol, Tween 80 and sterile saline (1:1:8). AM630 and AM251 were dissolved in dimethyl sulfoxide (DMSO, 99.7 %) and further diluted with vehicle. 45 min prior to the induction of TNBS colitis, mice were injected intraperitoneally (i.p.) with 5 mg/kg AM630, AM251, O-1918 or vehicle, followed by 5 mg/kg Abn-CBD, 15 min later. As a single dose of Abn-CBD was ineffective (preliminary data not shown), mice were injected twice daily for 3 days. For in vitro assays, 10 mM stock solutions (in DMSO) of Abn-CBD and the CB receptor antagonists were prepared.
Induction of TNBS colitis
The TNBS colitis experiment was used as an established model for testing cannabinoid effects on experimental colitis [4, 9, 16]. TNBS colitis was induced in male CD1 mice as described previously . Briefly, animals were lightly anaesthetized with isoflurane and TNBS (4 mg in 100 μL of 30 % ethanol) was infused into the colon through a catheter, 1 mm in diameter, inserted 3 cm proximally to the anus in mice. Vehicle alone (100 μL of 30 % ethanol) was administered in control experiments. The mice were weighed daily.
Macroscopic scoring and damage assessment
Four days after the induction of colitis, mice were euthanized by cervical dislocation. The colon was immediately removed, opened longitudinally along the mesenteric border, and examined. Colonic damage was assessed by a semi-quantitative scoring system as previously described . Macroscopic damage was scored according to the following scale, adding individual scores for ulcer, colonic shortening, wall thickness, and presence of hemorrhage, fecal blood, or diarrhea. Ulcer: 1 point for each 0.5 cm; shortening of the colon: 1 point = >15 %, 2 points = >25 % (based on a mean length of the untreated colon of 7.6 ± 0.55 cm; n = 5-10); wall thickness measured in mm. The presence of hemorrhage, fecal blood, or diarrhea increased the score by 1 point for each additional feature. Adhesion of the colon to organs was scored as follows: 1 point = 1 adhesion, 2 points = 2 or more adhesions or adhesions to organs. A new batch of TNBS was used for the experiments with the AM630 and AM251 inhibitors, likely being the reason for the higher overall damage score in these experiments.
Measurement of myeloperoxidase (MPO) activity in tissue samples was used to assess the degree of granulocyte infiltration . Samples of mouse colon were weighed, immediately frozen on dry ice, and stored at - 80 °C. For determination of MPO activity, the frozen tissue was placed in 0.5 % HTAB buffer (50 mg of tissue/mL; pH 6.0) and disrupted with a Polytron homogenizer (Brinkman Instruments, Mississauga, Ontario, Canada). The detergent HTAB (hexadecyl-trimethyl-ammoniumbromide; Sigma-Aldrich) releases MPO from the primary granules of neutrophils and enhances enzyme activity through the presence of bromide. Afterwards, the homogenate was centrifuged for 15 min at maximum speed and 4 °C. Before reading MPO activity, 7 μL of supernatant was added to 200 μL of 50 mM potassium phosphate buffer (pH 6.0) containing 0.167 mg/mL of O-dianisidine hydrochloride and 0.5 μL of 1 % H2O2/mL. The kinetics of MPO activity was measured at 460 nm (Thermo Fischer Labsystems Multiskan, Thermo Scientific, Ottawa, Ontario, Canada). A mean was calculated for the vehicle-treated group and set at 100 %. Values of all other treatment groups are expressed as percent of the respective vehicle-treated group (TNBS only).
For microscopic scoring, segments of the distal colon were stapled flat onto cardboard with the mucosal side up and fixed for 24 h in 10 % neutral-buffered formalin. Tissue was then dehydrated, embedded in paraffin, and standard hematoxylin/eosin staining was performed on 5-μm thick sections. Five sections at least 50 μm apart per colon were scored as described before . Mucosal architecture, muscle thickness, leukocyte infiltration were each scored with 0–3 points where 0 depicts normal colon and 3 the maximally affected colon. The absence of goblet cells scored 1 point. The total score index for one colon was the sum of these subscores and had a maximum of 9.
Neutrophil isolation and HUVEC culture
Neutrophils were isolated as previously described . Briefly, blood from healthy adult donors was drawn into a heparinized syringe, and allowed to settle on half of its volume of 6 % dextran for 1 h at room temperature. Erythrocytes were removed by hypotonic lysis, and granulocytes were further purified by density centrifugation on lymphoprep 1077. Endothelial cells (HUVEC) were isolated from human umbilical cords (Foothills Hospital, Calgary, Canada) as previously described  and maintained in M199 medium (Invitrogen, Carlsbad, CA) containing 20 % human serum. Only the first passage of cells was used for the experiments. All procedures requiring human subjects were approved by The University of Calgary Conjoint Health Research Ethics Board. Study participants provided written consent for donation of their biological samples for research. The colonic epithelial cell line LoVo was purchased from ATCC (# CCL-229) and cultured in F-12 K Medium (ATCC) supplemented with 10 % fetal bovine serum.
2.5 × 105 neutrophils were incubated with Abn-CBD and/or O-1918 at the concentrations indicated for 30 min and pipetted into the top chamber of 5 μm pore Transwells® (Corning Life Sciences, Union City, CA). Cells were allowed to migrate through the pores to the lower chamber of the wells containing the chemokine IL-8 at the effective concentration of 10nM. This suboptimal IL-8 concentration was chosen so that that anything that interfered with IL-8’s ability to induce a response could be observed. A random migration control (no stimulus) was run in every experiment. The experiment was stopped after 3 h. Three non-overlapping digital images of the bottom of each chamber were taken using a light microscope, and adherent cells were counted.
Neutrophil recruitment under flow conditions
Interactions between endothelial cells and freshly isolated human neutrophils were examined under flow conditions. A parallel plate flow chamber from Glycotech (Gaithersburg, MD, USA) was used to mimic physiologic flow conditions.
Neutrophil accumulation and transmigration were determined as previously described for eosinophils . Briefly, endothelial cell monolayers were incubated with 10 ng/ml of TNF-α for 4 h. Either the monolayer was also incubated with Abn-CBD or O-1918 for 4 h, or neutrophils were treated with the drugs 1 h prior to the experiment at indicated concentrations. After TNF-α stimulation, neutrophils (1 × 106/mL) were perfused across the monolayer at 1 dyn/cm2 using the parallel plate flow chamber. After 4 min of perfusion, the inlet line was transferred to Hank’s balanced salt solution (HBSS), to prevent the binding of new neutrophils, and buffer was perfused for an additional 4 min. Interactions between neutrophils and endothelial cells were visualized on a Zeiss Axiovert 100 microscope using either a 10X/0.25NA or 40X/0.60NA phase-contrast objective and recorded via a charge-coupled device camera (KP-M1U; Hitachi Denshi, Ltd.). The total number of cells accumulated on the monolayer was determined between 4 and 5 min of perfusion and the number of transmigrated cells were determined between 6 and 7 min. 4–10 fields of view were examined for each condition.
Neutrophils on glass coverslips were fixed with 2 % paraformaldehyde in HBSS, then washed with HBSS and blocked for 1 h with 1 % human serum albumin in HBSS. Cells were stained with anti-GPR18 antibody or isotype control for 1 h (1:300 in HBSS), then washed and incubated with the secondary FITC-conjugated detection antibody (1:1000) for 1 h. Cells were mounted with Hoechst 33258 (Sigma-Aldrich, Oakville, Ontario, Canada).
Scratch wound healing assay
Single-path wound healing experiments were performed as described . Briefly, LoVo cells and HUVEC were grown to confluence in 6-well dishes. A day prior to the experiment cells were starved with serum-deprived medium or medium with low serum content (for HUVEC). Wounds were made by dragging a sterile 10 μl pipette tip across the monolayer to create a cell-free path ~1 mm wide, and cells were incubated with Abn-CBD and/or O-1918 at the concentrations indicated for 20 h, or at 1 μM, where there is no indication. Pictures of the wounds at 0 h and 20 h were taken using an inverted microscope with mounted camera at x100 magnification (Olympus, Center Valley, PA), and quantification of cell migration into the wounded area was done using the MetaMorph® Analysis Software. 3-4 fields of view were analyzed per well.
For statistical analysis a Student’s t-test or one-way ANOVA with Tukey’s post-hoc test was performed using the GraphPad Prism software (La Jolla, CA, USA). Significance was set at p ≤ 0.05. Data are presented as mean values ± the standard error of the means (SEM).
Abn-CBD attenuates the degree of colitis
Protective effects of Abn-CBD in TNBS-induced colitis are CB1/CB2 independent
Abn-CBD inhibits inflammatory neutrophil recruitment
Abn-CBD promotes wound healing in endothelial and colonic epithelial cells
Endothelial and epithelial integrity is crucial for preserving vascular and intestinal homeostasis [30, 31]. IBD is characterized by ulceration and impairment of the intestinal epithelial barrier , and cannabinoids have been shown to promote wound healing of epithelial cells in a CB1 dependent manner . Therefore we tested if Abn-CBD could promote epithelial and endothelial wound healing.
The incidence and prevalence of ulcerative colitis and Crohn’s disease is increasing over time and in different regions of the world, suggesting that IBD is an emerging global disease . Numerous studies have revealed that endocannabinoid levels, and the expression of CB1 and CB2 receptors are increased in IBD patients and that the endocannabinoid system may play a protective role in the development of colitis [35, 36]. Moreover, other studies suggest that cannabinoids may exert anti-inflammatory effects in cardiovascular disease (reviewed in ), the number one cause of death globally . Therefore, cannabinoids that exert effects through the endocannabinoid system, but also through novel receptors may have therapeutic potential in inflammatory diseases, especially if the psychotropic properties can be eliminated.
In these studies we examined the therapeutic potential of the non-cannabinoid CBD analogue Abn-CBD for the treatment of inflammatory conditions of the gastrointestinal tract. Here we show that Abn-CBD treatment resulted in a 20–30 % reduction of TNBS-induced colitis in mice, hence Abn-CBD is less effective than previously studied cannabinoid receptor agonists and the atypical cannabinoid O-1602, which all reduced TNBS-induced colitis by 35–55 % [9, 17].
The beneficial effects of Abn-CBD were completely inhibited by O-1918, the antagonist for the putative Abn-CBD receptor, suggesting this was a receptor-mediated effect. Interestingly, O-1918 administration alone increased colitis scores and MPO activity beyond that of controls, which suggests that the actions of an endogenously active anti-inflammatory endocannabinoid were inhibited by O-1918. A previous study showed that O-1918 inhibits the activation of an “endothelial anandamide receptor”, and thus counteracting anandamide-induced vasorelaxation . It has also been demonstrated that anandamide is protective in experimental colitis, and that endogenous levels of anandamide are elevated in IBD patients . Therefore it seems likely that the administration of O-1918 not only blocked the effects of Abn-CBD, but also the effects of anandamide, causing more severe colon damage. We investigated the potential role of CB receptors in the mechanism of action of Abn-CBD. Neither CB1 nor CB2 receptor antagonists reversed the action of Abn-CBD when assessed at the macroscopic level. Previously it has been shown that CB2 receptor inhibition by AM630 exacerbates experimentally induced colitis . It seems that Abn-CBD treatment can compensate for the CB2 receptor inhibition by AM630, as the protective effect of Abn-CBD was not reduced by AM630 administration. The microscopic damage assessment revealed a slight inhibitory effect of AM251, which binds to CB1 and thereby inhibiting the receptor’s activation . AM251 has also been described to activate GPR55 , which is also a ligand for Abn-CBD, though Abn-CBD has a much higher EC50 for that receptor . Therefore, a subtle Abn-CBD effect conveyed by GPR55 may be antagonized by AM251.
In order to elucidate the effects of Abn-CBD we assessed wound healing and neutrophil recruitment. These experiments are important as endothelial dysfunction, delayed wound healing and neutrophil infiltration are key features of chronic inflammatory diseases, such as cardiovascular disease and IBD [24, 42–45]. Abn-CBD increased the wound healing capacity of HUVEC. This protective effect was slightly inhibited by the antagonist O-1918. Abn-CBD-induced wound healing of the colonic epithelial cell line LoVo was completely inhibited by O-1918, suggesting that Abn-CBD acts, at least partly through a different, O-1918-independent receptor in the endothelial cells.
In experiments to investigate the effects of Abn-CBD on neutrophil chemotaxis, IL-8 was used as neutrophil attractant, as this chemokine is highly expressed in the inflamed mucosa of IBD patients and also in uninflamed specimens from Crohn’s disease patients [46, 47]. The inhibitory effect of Abn-CBD on neutrophil chemotaxis was completely reversed by the antagonist O-1918 not only in the static chemotaxis assay, but also in a more physiologic neutrophil recruitment assay under flow conditions. Here, neutrophil activation and arrest triggered by inflamed endothelium was inhibited, when neutrophils were pre-treated with Abn-CBD, but not when the HUVEC monolayer was pre-treated with Abn-CBD. Abn-CBD had no significant effect on the pro-inflammatory actions of TNF-α on the endothelium. The process of neutrophil extravasation from the blood stream to sites of inflammation involves the rolling of the cells on the endothelium, firm arrest of the neutrophil, and transmigration through the endothelium. These particular steps require intricate overlapping and distinct molecular events to take place, such as activation of integrins, and increased expression of adhesion molecules and chemokines . In summary, rolling of neutrophils on the endothelium is selectin-mediated and followed by chemokine-triggered activation of leukocytes and their integrin-dependent arrest. Chemokines that lead to activation and adherence of neutrophils are produced by the inflamed endothelium and immobilized on the endothelial cell-surface . The last step of the extravasation process, the transmigration or diapedesis of the neutrophil through the endothelium, also involves redistribution of integrin-adhesion molecule clusters . As incubation of the HUVEC monolayer did not alter neutrophil accumulation, but treatment of neutrophils did, we reason that Abn-CBD interferes with the chemokine-mediated inside-out signaling in neutrophils through GPCRs, which would otherwise lead to an increase of affinity and avidity of integrins. This assumption concurs with the fact that neutrophil rolling (selectin-dependent) was not affected by Abn-CBD (data not shown), but transmigration (integrin-dependent) was. The identification of the inhibitory pathway and identification of the affected integrin-ligand interactions go beyond the scope of this study, but we speculate that Abn-CBD effects may be conferred by GPR18, the putative Abn-CBD receptor , as its expression on these cells was confirmed by immunohistochemistry. Furthermore the inhibition of neutrophil chemotaxis by other endocannabinoids were shown to be independent of CB1 and CB2 receptors .
This study demonstrates that Abn-CBD promotes epithelial, and endothelial wound healing in vitro. Abn-CBD drastically interferes with the inflammatory recruitment of neutrophils in vitro, and in vivo possibly through targeting the receptor. GPR18 is a strong candidate involved in the mechanism by which Abn-CBD improves the outcome of colitis, since attenuation of TNBS-induced colitis by Abn-CBD was not reversed by CB1/CB2 antagonists on a macroscopic level. These studies point to the potential of non-psychotropic cannabinoids as potential therapeutics for the treatment of IBD, but may also be of importance in other chronic inflammatory diseases like cardiovascular disease, as suggested by Abn-CBD’s strong effect on endothelial cells.
Abn-CBD, abnormal cannabidiol; AM251, N-(Piperidin-1-yl)-5-(4-iodophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide; AM630, 6-iodo-2-methyl-1-[2-(4-morpholinyl)ethyl-1H-indol-3-yl](4-methoxyphenyl)methanone; CB1, cannabinoid receptor 1; CB2, cannabinoid receptor 2; CBD, cannabidiol; DMSO, dimethyl sulfoxide; GPCR, G-protein-coupled receptor; IBD, inflammatory bowel disease; MPO, myeloperoxidase; O1918, 1,3-Dimethoxy-5-methyl-2-[(1R,6R)-3-methyl-6-(1-methylethenyl)-2-cyclohexen-1-yl]benz-ene; TNBS, trinitrobenzene sulfonic acid
We thank Catherine MacNaughton for skilled technical assistance. KAS is the Crohn’s Colitis Canada Chair in Inflammatory Bowel Disease Research.
This work was funded by Crohn’s and Colitis Canada (to MAS and KAS).
RMK designed and performed experiments, conducted analyses and wrote the manuscript. SAP conducted neutrophil recruitment experiments and analyses and critically reviewed the manuscript. KDP, RMY and JF designed flow chamber, wound healing and colitis experiments, respectively, and critically reviewed the manuscript. KAS and MAS designed the research, analysed data, contributed substantially to the manuscript and obtained funding for the studies. All authors read and approved the final manuscript.
The authors declare that they have no competing interests.
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