Endogenous cannabinoid system regulates intestinal barrier function in vivo through cannabinoid type 1 receptor activation

Abstract

Abstract The deleterious effects of stress on the gastrointestinal tract seem to be mainly mediated by the induction of intestinal barrier dysfunction and subsequent subtle mucosal inflammation. Cannabinoid 1 receptor (CB1R) is expressed in the mammalian gut under physiological circumstances. The aim of this investigation is to study the possible role of CB1R in the maintenance of mucosal homeostasis after stress exposure. CB1R knockout mice (CB1R−/−) and their wild-type (WT) counterparts were exposed to immobilization and acoustic (IA) stress for 2 h per day during 4 consecutive days. Colonic protein expression of the inducible forms of the nitric oxide synthase and cyclooxygenase (NOS2 and COX2), IgA production, permeability to 51Cr-EDTA, and bacterial translocation to mesenteric lymph nodes were evaluated. Stress exposure induced greater expression of proinflammatory enzymes NOS2 and COX2 in colonic mucosa of CB1R−/− mice when compared with WT animals. These changes were related with a greater degree of colonic barrier dysfunction in CB1R−/− animals determined by 1) a significantly lower IgA secretion, 2) higher paracellular permeability to 51Cr-EDTA, and 3) higher bacterial translocation, both under basal conditions and after IA stress exposure. Pharmacological antagonism with rimonabant reproduced stress-induced increase of proinflammatory enzymes in the colon described in CB1R−/− mice. In conclusion, CB1R exerts a protective role in the colon in vivo through the regulation of intestinal secretion of IgA and paracellular permeability. Pharmacological modulation of cannabinoid system within the gastrointestinal tract might be therapeutically useful in conditions on which intestinal inflammation and barrier dysfunction takes place after exposure to stress.

besides its essential digestive function, the gastrointestinal tract represents the main interplay between the host and the environment, exerting an effective but also selective barrier function between the gastrointestinal mucosal immune system and the virtually infinite microbial and alimentary antigens on the mucosal surface. Intestinal epithelial cells constitute the main element of this barrier and exert pivotal roles both in the generation of tolerance toward alimentary antigens and commensal microbiota, and in the activation and orchestration of effective innate and adaptive immune responses (7, 16, 46, 47). However, intestinal barrier is a dynamic structure constituted not only by cellular components but also by an array of noncellular elements such as mucin, antimicrobial peptides, secretory immunoglobulin A (IgA) as well as apical tight junctions between adjacent epithelial cells. Tight junctions are dynamic molecular structures that constitute the rate-limiting seal of the intestinal epithelial barrier paracellular pathway (30). A huge number of proteins take part in the structure of the tight junctions, including zonula occludens (ZO) family proteins, occludin, and the numerous proteins of the claudin family (53); furthermore, the junctional complex is closely related to a ring of actin microfilaments, contraction of which seems to directly regulate paracellular permeability.

Inflammatory cytokines such as interferon-γ and tumor necrosis factor-α are capable of regulating tight junction barrier function (31). Intestinal barrier dysfunction leads to the translocation to the lamina propria, lymphatic vessels and portal circulation of luminal bacteria capable of triggering and perpetuating local, and even systemic inflammation. It occurs, for example, in acute pancreatitis and advanced liver cirrhosis; moreover, an adequate transcellular absorption process depends on the presence of an intact tight junction barrier to maintain transepithelial concentration gradients. Indeed, increased intestinal permeability directly related to tight junction dysfunction is a characteristic feature of ulcerative colitis, Crohn's disease, celiac disease, and food allergies (2, 12). Also, intestinal tight junction disruption has been shown in experimental models of stress, assuming their direct responsibility on the increased intestinal permeability that characterizes acute stress in laboratory animals (11, 33, 37). In this sense, exposure to physical and psychological stress triggers and/or modifies the clinical course of a variety of gastrointestinal disorders such as irritable bowel syndrome (IBS) and inflammatory bowel diseases (IBD) (6, 49).

Growing evidence from experimental studies supports the ability of psychosocial stress to induce biochemical and histological inflammatory changes in the intestinal mucosa. Indeed, animal stress models represent an excellent tool to assess intestinal barrier physiology and pathophysiology. A common finding observed in several models of stress-induced intestinal inflammation is an increased expression and activity of the inducible isoforms of nitric oxide synthase (NOS2) and cyclooxygenase (COX2) in intestinal tissue homogenates (9). The resulting high concentrations of NO and other reactive oxygen and nitrogen species produced by NOS2 as well as COX2-derived prostaglandin E2 have been involved in barrier dysfunction (3) and water/chloride secretion (48), respectively, associated with intestinal inflammatory conditions. A wide variety of results suggest that such intestinal inflammatory response triggered by psychological and physical stress could be mediated, at least in part, by the induction of intestinal barrier dysfunction resulting in bacterial translocation and enhanced uptake of luminal antigens (9).

The endogenous cannabinoid system regulates many different functions in the gastrointestinal system of vertebrates. The two types of cannabinoid receptors (CBR) that have been discovered and cloned, CB1R and CB2R (20), are differentially expressed in the human colon; whereas CB1R is expressed in intestinal epithelial cells, smooth muscle, myenteric plexus, and lamina propria plasma cells, CB2R is mainly expressed under physiological circumstances in plasma cells and macrophages (51) but has also been recently found in myenteric and submucosal neurons of rodent (13) and human bowel samples (52). The effects of CBR activation and the physiological roles for endocannabinoids in the gastrointestinal tract have been extensively reviewed (23); briefly, CB1R activation acts mostly via brain-gut axis to reduce gastrointestinal motility, diarrhea, pain or hyperalgesia, transient lower esophageal sphincter relaxations, emesis, and gastric acid secretion, as well as to promote eating; CB2R activation acts mostly via immune cells to reduce inflammation (39) through, at least, an inhibitory effect on interleukin-8 release in human colonic epithelial cells (21). A role for gastrointestinal carcinogenesis has also been suggested, as downregulation of CB1R and upregulation of CB2R have been observed in intestinal samples of patients with colon cancer (22). However, the role of the endocannabinoid system, and in particular CB1R, in intestinal barrier function and mucosal homeostasis is still largely unknown; however, although the exact mechanisms are poorly understood, some findings support the notion of an endogenous anti-inflammatory activity of CB1R because mice lacking CB1R show enhanced colitis compared with their wild-type (WT) littermates (32, 42). Consistent with this observation, administration of CBR agonists (26) or targeting endocannabinoid degradation (43) has been shown to protect against various forms of experimental colitis in animal models. Nevertheless, the role of the endocannabinoid system in intestinal barrier function has not been previously explored.

Therefore, in the present study, we aim to investigate whether CB1R modulates intestinal barrier function in mice exposed to immobilization and acoustic (IA) stress; for this purpose we took advantage of the use of genetically modified mice lacking CB1R as well as pharmacological manipulation of CB1R. We report herein that stress-induced changes were related with a greater colonic barrier permeability and inflammation, lower IgA secretion, and higher bacterial translocation when CB1R is absent.