Proteolipid protein 1 is involved in the regulation of intestinal motility and barrier function in the mouse.

Proteolipid protein 1 (Plp1) is highly expressed in enteric glia, labeling cells throughout the mucosa, muscularis, and the extrinsic innervation. Plp1 is a major constituent of myelin in the central and peripheral nervous systems, but the absence of myelin in the enteric nervous system (ENS) suggests another role for Plp1 in the gut. Although the functions of enteric glia are still being established, there is strong evidence that they regulate intestinal motility and permeability. To interrogate the role of Plp1 in enteric glia, we investigated gut motility, secretomotor function and permeability, and evaluated the ENS in mice lacking Plp1. We studied two time points: ∼3 mo (young) and >1 yr (old). Old Plp1 null mice exhibited increased fecal output, decreased fecal water content, faster whole gut transit times, reduced intestinal permeability, and faster colonic migrating motor complexes. Interestingly, in both young and old mice, the ENS exhibited normal glial and neuronal numbers as well as glial arborization density in the absence of Plp1. As Plp1-associated functions involve mitogen-activated protein kinase/extracellular signal-regulated kinase 1/2 (Mapk/Erk1/2) signaling and Mapk/Erk1/2 are reported to have a regulatory role in intestinal motility, we measured protein expression of Erk1/2 and its active form in the small intestine. Old Plp1 null mice had reduced levels of phosphorylated-Erk1/2. Although Plp1 is not required for the normal appearance of enteric glial cells, it has a regulatory role in intestinal motility and barrier function. Our results suggest that functional changes mediated by Plp1-expressing enteric glia may involve Erk1/2 activation.NEW & NOTEWORTHY Here, we describe that Plp1 regulates gut motility and barrier function. The functional effects of Plp1 eradication are only seen in old mice, not young. The effects of Plp1 appear to be mediated through the Erk1/2 pathway.

A monocyte-leptin-angiogenesis pathway critical for repair post-infection.

During infection, inflammatory monocytes are thought to be key for bacterial eradication, but this is hard to reconcile with the large numbers of neutrophils that are recruited for each monocyte that migrates to the afflicted tissue, and the much more robust microbicidal functions of the neutrophils. However, unlike neutrophils, monocytes have the capacity to convert to situationally specific macrophages that may have critical functions beyond infection control1,2. Here, using a foreign body coated with Staphylococcus aureus and imaging over time from cutaneous infection to wound resolution, we show that monocytes and neutrophils are recruited in similar numbers with low-dose infection but not with high-dose infection, and form a localization pattern in which monocytes surround the infection site, whereas neutrophils infiltrate it. Monocytes did not contribute to bacterial clearance but converted to macrophages that persisted for weeks after infection, regulating hypodermal adipocyte expansion and production of the adipokine hormone leptin. In infected monocyte-deficient mice there was increased persistent hypodermis thickening and an elevated leptin level, which drove overgrowth of dysfunctional blood vasculature and delayed healing, with a thickened scar. Ghrelin, which opposes leptin function3, was produced locally by monocytes, and reduced vascular overgrowth and improved healing post-infection. In sum, we find that monocytes function as a cellular rheostat by regulating leptin levels and revascularization during wound repair.

Role of CB1 receptors in the acute regulation of small intestinal permeability: effects of high-fat diet.

The endocannabinoid system of the gastrointestinal tract is involved in the control of intestinal barrier function. Whether the cannabinoid 1 (CB1) receptor is expressed on the intestinal epithelium and acutely regulates barrier function has not been determined. Here, we tested the hypothesis that ligands of the CB1 receptor acutely modulate small intestinal permeability and that this is associated with altered distribution of tight junction proteins. We examined the acute effects of CB1 receptor ligands on small intestinal permeability both in chow-fed and 2-wk high-fat diet (HFD)-fed mice using Ussing chambers. We assessed the distribution of CB1 receptor and tight junction proteins using immunofluorescence and the expression of CB1 receptor using PCR. A low level of CB1 expression was found on the intestinal epithelium. CB1 receptor was highly expressed on enteric nerves in the lamina propria. Neither the CB1/CB2 agonist CP55,940 nor the CB1 neutral antagonist AM6545 altered the flux of 4kDa FITC dextran (FD4) across the jejunum or ileum of chow-fed mice. Remarkably, both CP55,940 and AM6545 reduced FD4 flux across the jejunum and ileum in HFD-fed mice that have elevated baseline intestinal permeability. These effects were absent in CB1 knockout mice. CP55,940 reduced the expression of claudin-2, whereas AM6545 had little effect on claudin-2 expression. Neither ligand altered the expression of ZO-1. Our data suggest that CB1 receptor on the intestinal epithelium regulates tight junction protein expression and restores barrier function when it is increased following exposure to a HFD for 2 wk.NEW & NOTEWORTHY The endocannabinoid system of the gastrointestinal tract regulates homeostasis by acting as brake on motility and secretion. Here we show that when exposed to a high fat diet, intestinal permeability is increased and activation of the CB1 receptor on the intestinal epithelium restores barrier function. This work further highlights the role of the endocannabinoid system in regulating intestinal homeostasis when it is perturbed.

Genetic Variants of Fatty Acid Amide Hydrolase Modulate Acute Inflammatory Responses to Colitis in Adult Male Mice.

Cannabinoids, including cannabis derived phytocannabinoids and endogenous cannabinoids (endocannabinoids), are typically considered anti-inflammatory. One such endocannabinoid is N-arachidonoylethanolamine (anandamide, AEA), which is metabolized by fatty acid amide hydrolase (FAAH). In humans, there is a loss of function single nucleotide polymorphism (SNP) in the FAAH gene (C385A, rs324420), that leads to increases in the levels of AEA. Using a mouse model with this SNP, we investigated how this SNP affects inflammation in a model of inflammatory bowel disease. We administered 2,4,6-trinitrobenzene sulfonic acid (TNBS) intracolonically, to adult male FAAH SNP mice and examined colonic macroscopic tissue damage and myeloperoxidase activity, as well as levels of plasma and amygdalar cytokines and chemokines 3 days after administration, at the peak of colitis. We found that mice possessing the loss of function alleles (AC and AA), displayed no differences in colonic damage or myeloperoxidase activity compared to mice with wild type alleles (CC). In contrast, in plasma, colitis-induced increases in interleukin (IL)-2, leukemia inhibitory factor (LIF), monocyte chemoattractant protein (MCP)-1, and tumor necrosis factor (TNF) were reduced in animals with an A allele. A similar pattern was observed in the amygdala for granulocyte colony stimulating factor (G-CSF) and MCP-1. In the amygdala, the mutant A allele led to lower levels of IL-1α, IL-9, macrophage inflammatory protein (MIP)-1ß, and MIP-2 independent of colitis-providing additional understanding of how FAAH may serve as a regulator of inflammatory responses in the brain. Together, these data provide insights into how FAAH regulates inflammatory processes in disease.

Intestinal microbiota shapes gut physiology and regulates enteric neurons and glia.

BACKGROUND: The intestinal microbiota plays an important role in regulating gastrointestinal (GI) physiology in part through interactions with the enteric nervous system (ENS). Alterations in the gut microbiome frequently occur together with disturbances in enteric neural control in pathophysiological conditions. However, the mechanisms by which the microbiota regulates GI function and the structure of the ENS are incompletely understood. Using a mouse model of antibiotic (Abx)-induced bacterial depletion, we sought to determine the molecular mechanisms of microbial regulation of intestinal function and the integrity of the ENS. Spontaneous reconstitution of the Abx-depleted microbiota was used to assess the plasticity of structure and function of the GI tract and ENS. Microbiota-dependent molecular mechanisms of ENS neuronal survival and neurogenesis were also assessed. RESULTS: Adult male and female Abx-treated mice exhibited alterations in GI structure and function, including a longer small intestine, slower transit time, increased carbachol-stimulated ion secretion, and increased intestinal permeability. These alterations were accompanied by the loss of enteric neurons in the ileum and proximal colon in both submucosal and myenteric plexuses. A reduction in the number of enteric glia was only observed in the ileal myenteric plexus. Recovery of the microbiota restored intestinal function and stimulated enteric neurogenesis leading to increases in the number of enteric glia and neurons. Lipopolysaccharide (LPS) supplementation enhanced neuronal survival alongside bacterial depletion, but had no effect on neuronal recovery once the Abx-induced neuronal loss was established. In contrast, short-chain fatty acids (SCFA) were able to restore neuronal numbers after Abx-induced neuronal loss, demonstrating that SCFA stimulate enteric neurogenesis in vivo. CONCLUSIONS: Our results demonstrate a role for the gut microbiota in regulating the structure and function of the GI tract in a sex-independent manner. Moreover, the microbiota is essential for the maintenance of ENS integrity, by regulating enteric neuronal survival and promoting neurogenesis. Molecular determinants of the microbiota, LPS and SCFA, regulate enteric neuronal survival, while SCFA also stimulates neurogenesis. Our data reveal new insights into the role of the gut microbiota that could lead to therapeutic developments for the treatment of enteric neuropathies. Video abstract.

Novel Functionalized Cannabinoid Receptor Probes: Development of Exceptionally Potent Agonists.

We report the development of novel cannabinergic probes that can stabilize the cannabinoid receptors (CBRs) through tight binding interactions. Ligand design involves the introduction of select groups at a judiciously chosen position within the classical hexahydrocannabinol template (monofunctionalized probes). Such groups include the electrophilic isothiocyanato, the photoactivatable azido, and the polar cyano moieties. These groups can also be combined to produce bifunctionalized probes potentially capable of interacting at two distinct sites within the CBR-binding domains. These novel compounds display remarkably high binding affinities for CBRs and are exceptionally potent agonists. A key ligand (27a, AM11245) exhibits exceptionally high potency in both in vitro and in vivo assays and was designated as "megagonist," a property attributed to its tight binding profile. By acting both centrally and peripherally, 27a distinguishes itself from our previously reported "megagonist" AM841, whose functions are restricted to the periphery.

Comorbid anxiety-like behavior in a rat model of colitis is mediated by an upregulation of corticolimbic fatty acid amide hydrolase.

Peripheral inflammatory conditions, including those localized to the gastrointestinal tract, are highly comorbid with psychiatric disorders such as anxiety and depression. These behavioral symptoms are poorly managed by conventional treatments for inflammatory diseases and contribute to quality of life impairments. Peripheral inflammation is associated with sustained elevations in circulating glucocorticoid hormones, which can modulate central processes, including those involved in the regulation of emotional behavior. The endocannabinoid (eCB) system is exquisitely sensitive to these hormonal changes and is a significant regulator of emotional behavior. The impact of peripheral inflammation on central eCB function, and whether this is related to the development of these behavioral comorbidities remains to be determined. To examine this, we employed the trinitrobenzene sulfonic acid-induced model of colonic inflammation (colitis) in adult, male, Sprague Dawley rats to produce sustained peripheral inflammation. Colitis produced increases in behavioral measures of anxiety and elevations in circulating corticosterone. These alterations were accompanied by elevated hydrolytic activity of the enzyme fatty acid amide hydrolase (FAAH), which hydrolyzes the eCB anandamide (AEA), throughout multiple corticolimbic brain regions. This elevation of FAAH activity was associated with broad reductions in the content of AEA, whose decline was driven by central corticotropin releasing factor type 1 receptor signaling. Colitis-induced anxiety was reversed following acute central inhibition of FAAH, suggesting that the reductions in AEA produced by colitis contributed to the generation of anxiety. These data provide a novel perspective for the pharmacological management of psychiatric comorbidities of chronic inflammatory conditions through modulation of eCB signaling.

Intestinal fungi are causally implicated in microbiome assembly and immune development in mice.

The gut microbiome consists of a multi-kingdom microbial community. Whilst the role of bacteria as causal contributors governing host physiological development is well established, the role of fungi remains to be determined. Here, we use germ-free mice colonized with defined species of bacteria, fungi, or both to differentiate the causal role of fungi on microbiome assembly, immune development, susceptibility to colitis, and airway inflammation. Fungal colonization promotes major shifts in bacterial microbiome ecology, and has an independent effect on innate and adaptive immune development in young mice. While exclusive fungal colonization is insufficient to elicit overt dextran sulfate sodium-induced colitis, bacterial and fungal co-colonization increase colonic inflammation. Ovalbumin-induced airway inflammation reveals that bacterial, but not fungal colonization is necessary to decrease airway inflammation, yet fungi selectively promotes macrophage infiltration in the airway. Together, our findings demonstrate a causal role for fungi in microbial ecology and host immune functionality, and therefore prompt the inclusion of fungi in therapeutic approaches aimed at modulating early life microbiomes.

Endocannabinoid regulation of homeostatic feeding and stress-induced alterations in food intake in male rats.

BACKGROUND AND PURPOSE: Stress is known to reduce food intake. Many aspects of the stress response and feeding are regulated by the endocannabinoid system, but the roles of anandamide (AEA) and 2-arachidonoyl glycerol (2-AG) in stress-induced anorexia are unclear. EXPERIMENTAL APPROACH: Effects of acute restraint stress on endocannabinoids were investigated in male Sprague-Dawley rats. Systemic and central pharmacological inhibition of fatty acid amide hydrolase (FAAH) or monoacylglycerol lipase (MAGL) was used to assess the effects of elevated AEA and 2-AG on homeostatic feeding and on food consumption after stress. Animals were pretreated with the FAAH inhibitor, PF-04457845, or the MAGL inhibitor, MJN110, before 2 h acute restraint stress or 2 h homecage period without food. KEY RESULTS: Restraint stress decreased hypothalamic and circulating AEA, with no effect in the gastrointestinal tract, while 2-AG content in the jejunum (but not duodenum) was reduced. PF-04457845 (30 µg), given i.c.v., attenuated stress-induced anorexia via CB1 receptors, but reduced homeostatic feeding in unstressed animals through an unknown mechanism. On the other hand, systemic administration of MJN110 (10 mg·kg-1 ) reduced feeding, regardless of stress or feeding status and inhibited basal intestinal transit in unstressed rats. The ability of MAGL inhibition to reduce feeding in combination with stress was independent of CB1 receptor signalling in the gut as the peripherally restricted CB1 receptor antagonist, AM6545 did not block this effect. CONCLUSIONS AND IMPLICATIONS: Our data reveal diverse roles for 2-AG and AEA in homeostatic feeding and changes in energy intake following stress. LINKED ARTICLES: This article is part of a themed section on 8th European Workshop on Cannabinoid Research. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v176.10/issuetoc.

Alterations in melatonin and 5-HT signalling in the colonic mucosa of mice with dextran-sodium sulfate-induced colitis.

BACKGROUND AND PURPOSE: Inflammatory bowel disease (IBD) is characterized by pain, bleeding, cramping and altered gastrointestinal (GI) function. Changes in mucosal 5-HT (serotonin) signalling occur in animal models of colitis and in humans suffering from IBD. Melatonin is co-released with 5-HT from the mucosa and has a wide variety of actions in the GI tract. Here, we examined how melatonin signalling is affected by colitis and determined how this relates to 5-HT signalling. EXPERIMENTAL APPROACH: Using electroanalytical approaches, we investigated how 5-HT release, reuptake and availability as well as melatonin availability are altered in dextran sodium sulfate (DSS)-induced colitis in mice. Studies were conducted to explore if melatonin treatment during active colitis could reduce the severity of colitis. KEY RESULTS: We observed an increase in 5-HT and a decrease in melatonin availability in DSS-induced colitis. A significant reduction in 5-HT reuptake was observed in DSS-induced colitis animals. A reduction in the content of 5-HT was observed, but no difference in tryptophan levels were observed. A reduction in deoxycholic acid-stimulated 5-HT availability and a significant reduction in mechanically-stimulated 5-HT and melatonin availability were observed in DSS-induced colitis. Orally or rectally administered melatonin once colitis was established did not significantly suppress inflammation. CONCLUSION AND IMPLICATIONS: Our data suggest that DSS-induced colitis results in a reduction in melatonin availability and an increase in 5-HT availability, due to a reduction/loss of tryptophan hydroxylase 1 enzyme, 5-HT content and 5-HT transporters. Mechanosensory release was more susceptible to inflammation when compared with chemosensory release.

Protective Actions of Epithelial 5-Hydroxytryptamine 4 Receptors in Normal and Inflamed Colon.

BACKGROUND & AIMS: The 5-hydroxytryptamine receptor 4 (5-HT4R or HTR4) is expressed in the colonic epithelium but little is known about its functions there. We examined whether activation of colonic epithelial 5-HT4R protects colons of mice from inflammation. METHODS: The 5-HT4R agonist tegaserod (1 mg/kg), the 5-HT4R antagonist GR113808 (1 mg/kg), or vehicle (control) were delivered by enema to wild-type or 5-HT4R knockout mice at the onset of, or during, active colitis, induced by administration of dextran sodium sulfate or trinitrobenzene sulfonic acid. Inflammation was measured using the colitis disease activity index and by histologic analysis of intestinal tissues. Epithelial proliferation, wound healing, and resistance to oxidative stress-induced apoptosis were assessed, as was colonic motility. RESULTS: Rectal administration of tegaserod reduced the severity of colitis compared with mice given vehicle, and accelerated recovery from active colitis. Rectal tegaserod did not improve colitis in 5-HT4R knockout mice, and intraperitoneally administered tegaserod did not protect wild-type mice from colitis. Tegaserod increased proliferation of crypt epithelial cells. Stimulation of 5-HT4R increased Caco-2 cell migration and reduced oxidative stress-induced apoptosis; these actions were blocked by co-administration of the 5-HT4R antagonist GR113808. In noninflamed colons of wild-type mice not receiving tegaserod, inhibition of 5-HT4Rs resulted in signs of colitis within 3 days. In these mice, epithelial proliferation decreased and bacterial translocation to the liver and spleen was detected. Daily administration of tegaserod increased motility in inflamed colons of guinea pigs and mice, whereas administration of GR113808 disrupted motility in animals without colitis. CONCLUSIONS: 5-HT4R activation maintains motility in healthy colons of mice and guinea pigs, and reduces inflammation in colons of mice with colitis. Agonists might be developed as treatments for patients with inflammatory bowel diseases.

Prevention of Diet-Induced Obesity Effects on Body Weight and Gut Microbiota in Mice Treated Chronically with Δ9-Tetrahydrocannabinol.

OBJECTIVE: Acute administration of cannabinoid CB1 receptor agonists, or the ingestion of cannabis, induces short-term hyperphagia. However, the incidence of obesity is lower in frequent cannabis users compared to non-users. Gut microbiota affects host metabolism and altered microbial profiles are observed in obese states. Gut microbiota modifies adipogenesis through actions on the endocannabinoid system. This study investigated the effect of chronic THC administration on body weight and gut microbiota in diet-induced obese (DIO) and lean mice. METHODS: Adult male DIO and lean mice were treated daily with vehicle or THC (2mg/kg for 3 weeks and 4 mg/kg for 1 additional week). Body weight, fat mass, energy intake, locomotor activity, whole gut transit and gut microbiota were measured longitudinally. RESULTS: THC reduced weight gain, fat mass gain and energy intake in DIO but not lean mice. DIO-induced changes in select gut microbiota were prevented in mice chronically administered THC. THC had no effect on locomotor activity or whole gut transit in either lean or DIO mice. CONCLUSIONS: Chronic THC treatment reduced energy intake and prevented high fat diet-induced increases in body weight and adiposity; effects that were unlikely to be a result of sedation or altered gastrointestinal transit. Changes in gut microbiota potentially contribute to chronic THC-induced actions on body weight in obesity.

Inhibiting Inducible Nitric Oxide Synthase in Enteric Glia Restores Electrogenic Ion Transport in Mice With Colitis.

BACKGROUND & AIMS: Disturbances in the control of ion transport lead to epithelial barrier dysfunction in patients with colitis. Enteric glia regulate intestinal barrier function and colonic ion transport. However, it is not clear whether enteric glia are involved in epithelial hyporesponsiveness. We investigated enteric glial regulation of ion transport in mice with trinitrobenzene sulfonic acid- or dextran sodium sulfate-induced colitis and in Il10(-/-) mice. METHODS: Electrically evoked ion transport was measured in full-thickness segments of colon from CD1 and Il10(-/-) mice with or without colitis in Ussing chambers. Nitric oxide (NO) production was assessed using amperometry. Bacterial translocation was investigated in the liver, spleen, and blood of mice. RESULTS: Electrical stimulation of the colon evoked a tetrodotoxin-sensitive chloride secretion. In mice with colitis, ion transport almost completely disappeared. Inhibiting inducible NO synthase (NOS2), but not neuronal NOS (NOS1), partially restored the evoked secretory response. Blocking glial function with fluoroacetate, which is not a NOS2 inhibitor, also partially restored ion transport. Combined NOS2 inhibition and fluoroacetate administration fully restored secretion. Epithelial responsiveness to vasoactive intestinal peptide was increased after enteric glial function was blocked in mice with colitis. In colons of mice without colitis, NO was produced in the myenteric plexus almost completely via NOS1. NO production was increased in mice with colitis, compared with mice without colitis; a substantial proportion of NOS2 was blocked by fluoroacetate administration. Inhibition of enteric glial function in vivo reduced the severity of trinitrobenzene sulfonic acid-induced colitis and associated bacterial translocation. CONCLUSIONS: Increased production of NOS2 in enteric glia contributes to the dysregulation of intestinal ion transport in mice with colitis. Blocking enteric glial function in these mice restores epithelial barrier function and reduces bacterial translocation.

Interactive effects of oligofructose and obesity predisposition on gut hormones and microbiota in diet-induced obese rats.

OBJECTIVE: Oligofructose (OFS) is a prebiotic that reduces energy intake and fat mass via changes in gut satiety hormones and microbiota. The effects of OFS may vary depending on predisposition to obesity. The aim of this study was to examine the effect of OFS in diet-induced obese (DIO) and diet-resistant (DR) rats. METHODS: Adult, male DIO, and DR rats were randomized to: high-fat/high-sucrose (HFS) diet or HFS diet + 10% OFS for 6 weeks. Body composition, food intake, gut microbiota, plasma gut hormones, and cannabinoid CB(1) receptor expression in the nodose ganglia were measured. RESULTS: OFS reduced body weight, energy intake, and fat mass in both phenotypes (P < 0.05). Select gut microbiota differed in DIO versus DR rats (P < 0.05), the differences being eliminated by OFS. OFS did not modify plasma ghrelin or CB(1) expression in nodose ganglia, but plasma levels of GIP were reduced and PYY were elevated (P < 0.05) by OFS. CONCLUSIONS: OFS was able to reduce body weight and adiposity in both prone and resistant obese phenotypes. OFS-induced changes in gut microbiota profiles in DIO and DR rats, along with changes in gut hormone levels, likely contribute to the sustained lower body weights.

Substrate-Selective Inhibition of Cyclooxygenase-2: Development and Evaluation of Achiral Profen Probes.

Cyclooxygenase-2 (COX-2) oxygenates arachidonic acid and the endocannabinoids 2-arachidonoylglycerol (2-AG) and arachidonoylethanolamide (AEA). We recently reported that (R)-profens selectively inhibit endocannabinoid oxygenation but not arachidonic acid oxygenation. In this work, we synthesized achiral derivatives of five profen scaffolds and evaluated them for substrate-selective inhibition using in vitro and cellular assays. The size of the substituents dictated the inhibitory strength of the analogs, with smaller substituents enabling greater potency but less selectivity. Inhibitors based on the flurbiprofen scaffold possessed the greatest potency and selectivity, with desmethylflurbiprofen (3a) exhibiting an IC(50) of 0.11 µM for inhibition of 2-AG oxygenation. The crystal structure of desmethylflurbiprofen complexed to mCOX-2 demonstrated a similar binding mode to other profens. Desmethylflurbiprofen exhibited a half-life in mice comparable to that of ibuprofen. The data presented suggest that achiral profens can act as lead molecules toward in vivo probes of substrate-selective COX-2 inhibition.

Endogenous prion protein attenuates experimentally induced colitis.

Although the cellular prion protein (PrP(C)) is expressed in the enteric nervous system and lamina propria, its function(s) in the gut is unknown. Because PrP(C) may exert a cytoprotective effect in response to various physiologic stressors, we hypothesized that PrP(C) expression levels might modulate the severity of experimental colitis. We evaluated the course of dextran sodium sulfate (DSS)-induced colitis in hemizygous Tga20 transgenic mice (approximately sevenfold overexpression of PrP(C)), Prnp(-/-) mice, and wild-type mice. On day 7, colon length, disease severity, and histologic activity indices were determined. Unlike DSS-treated wild-type and Prnp(-/-) animals, PrP(C) overexpressing mice were resistant to colitis induction, exhibited much milder histopathologic features, and did not exhibit weight loss or colonic shortening. In keeping with these results, pro-survival molecule expression and/or phosphorylation levels were elevated in DSS-treated Tga20 mice, whereas pro-inflammatory cytokine production and pSTAT3 levels were reduced. In contrast, DSS-treated Prnp(-/-) mice exhibited increased BAD protein expression and a cytokine expression profile predicted to favor inflammation and differentiation. PrP(C) expression from both the endogenous Prnp locus or the Tga20 transgene was increased in the colons of DSS-treated mice. Considered together, these findings demonstrate that PrP(C) has a previously unrecognized cytoprotective and/or anti-inflammatory function within the murine colon.

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.

The endocannabinoid system is involved in the regulation of gastrointestinal (GI) motility and inflammation. Using the peripherally restricted cannabinoid (CB)(1)/CB(2) receptor agonist naphthalen-1-yl-(4-pentyloxynaphthalen-1-yl)methanone (SAB378), we investigated the role of peripheral cannabinoid receptors in the regulation of GI motility and the development of colitis in mice. The actions of SAB378 on whole gut transit, upper GI transit, colonic propulsion, and locomotor activity were investigated in C57BL/6N, CB(1) receptor knockout, and CB(2) receptor knockout mice. The potential for SAB378 to modify inflammation was studied by using dextran sulfate sodium (DSS) and 2,4,6-trinitrobenzene sulfonic acid (TNBS) models of experimental colitis. SAB378 did not modify locomotor activity. SAB378 slowed all parameters of GI motility, and these effects were significantly reduced by the CB(1) receptor antagonist N-(piperidin-1-yl)-5-(4-iodophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3 carboxamide (AM251), but not by the CB(2) receptor antagonist 6-iodo-2-methyl-1-[2-(4-morpholinyl)ethyl]-1H- indol-3-yl](4-methoxyphenyl)methanone (AM630). SAB378 did not inhibit GI transit or colonic propulsion in CB(1) receptor knockout mice, whereas its effects were observed in CB(2) receptor knockout mice. SAB378 did not reduce the degree of colitis induced by DSS or TNBS. The actions of SAB378 on GI motility are mediated by peripherally located CB(1) receptors. SAB378 was not effective against two models of experimental colitis, which may indicate that peripheral cannabinoid receptor stimulation alone may not be sufficient to mediate the anti-inflammatory effects of cannabinoids.

Activation of the cannabinoid 2 receptor (CB2) protects against experimental colitis.

BACKGROUND: Activation of cannabinoid (CB)(1) receptors results in attenuation of experimental colitis. Our aim was to examine the role of CB(2) receptors in experimental colitis using agonists (JWH133, AM1241) and an antagonist (AM630) in trinitrobenzene sulfonic acid (TNBS)-induced colitis in wildtype and CB(2) receptor-deficient (CB(2) (-/-)) mice. METHODS: Mice were treated with TNBS to induce colitis and then given intraperitoneal injections of the CB(2) receptor agonists JWH133, AM1241, or the CB(2) receptor antagonist AM630. Additionally, CB(2) (-/-) mice were treated with TNBS and injected with JWH133 or AM1241. Animals were examined 3 days after the induction of colitis. The colons were removed for macroscopic and microscopic evaluation, as well as the determination of myeloperoxidase activity. Quantitative reverse-transcriptase polymerase chain reaction (RT-PCR) for CB(2) receptor was also performed in animals with TNBS and dextran sodium sulfate colitis. RESULTS: Intracolonic installation of TNBS caused severe colitis. CB(2) mRNA expression was significantly increased during the course of experimental colitis. Three-day treatment with JWH133 or AM1241 significantly reduced colitis; AM630 exacerbated colitis. The effect of JWH133 was abolished when animals were pretreated with AM630. Neither JWH133 nor AM1241 had effects in CB(2) (-/-) mice. CONCLUSIONS: We show that activation of the CB(2) receptor protects against experimental colitis in mice. Increased expression of CB(2) receptor mRNA and aggravation of colitis by AM630 suggests a role for this receptor in normally limiting the development of colitis. These results support the idea that the CB(2) receptor may be a possible novel therapeutic target in inflammatory bowel disease.

Differential adipokine response in genetically predisposed lean and obese rats during inflammation: a role in modulating experimental colitis?

The relationship between a predisposition to obesity and the development of colitis is not well understood. Our aim was to characterize the adipokine response and the extent of colitis in diet-induced obese (DIO) rats. DIO and control, diet-resistant (DR) animals were administered either saline or trinitrobenzene sulfonic acid (TNBS) to induce colitis. Macroscopic damage scores and myeloperoxidase (MPO) activity were measured to determine the extent of inflammation. Trunk blood was collected for the analysis of plasminogen activator inhibitor-1 (PAI-1) as well as leptin, ghrelin, and adiponectin. Colonic epithelial physiology was assessed using Ussing chambers. DIO rats had a modestly increased circulating PAI-1 before TNBS treatment; however, during colitis, DR animals had more than a fourfold increase in circulating PAI-1 compared with DIO rats. Circulating leptin was higher in DIO rats compared with DR animals, in the inflamed and noninflamed states. These changes in TNBS-induced adipokine profile were accompanied by decreased macroscopic tissue damage score in DIO animals compared with DR tissues. Furthermore, TNBS-treated DR animals lost significantly more weight than DIO rats during active inflammation. Colonic epithelial physiology was comparable between groups, as was MPO activity. The factors contributing to the decreased colonic damage are almost certainly multifold, driven by both genetic and environmental factors, of which adipokines are likely to play a part given the increasing body of evidence for their role in modulating intestinal inflammation.

Neuromuscular changes in a rat model of colitis.

Intracolonic administration of Trichinella spiralis larvae in rats causes colitis with features similar to ulcerative colitis, notably with inflammation predominantly limited to the colonic mucosa. Our aim was to characterize the functional and neurochemical changes occurring within the myenteric (MP) and submucosal plexuses (SMP) during T. spiralis-induced colitis. Infected rats had decreased body weight, altered stool consistency and elevated myeloperoxidase activity, 6 and 14 days post-infection (PI). Responses to acetylcholine and KCl in circular muscle strips were reduced in infected tissues, demonstrating an impairment of contractility. In addition, there was a decrease in spontaneous motor activity and reduced sensitivity to the nitric oxide synthase (NOS) inhibitor L-NOArg, corresponding with a significant reduction in NOS immunoreactive neurons in the MP of infected animals. T. spiralis did not alter the total number of myenteric or submucosal neurons. Substance P innervation of submucosal blood vessels was reduced after infection, as were submucosal calretinin and calbindin immunoreactive neurons. No changes in choline acetyltransferase and calcitonin gene-related peptide immunoreactivity were observed. T. spiralis-induced colitis causes profound neuromuscular adaptations. The reduction in NOS neurons appears to underlie changes in motility.