The endogenous cannabinoid system protects against colonic inflammation

Abstract

Excessive inflammatory responses can emerge as a potential danger for organisms' health. Physiological balance between pro- and anti-inflammatory processes constitutes an important feature of responses against harmful events. Here, we show that cannabinoid receptors type 1 (CB1) mediate intrinsic protective signals that counteract proinflammatory responses. Both intrarectal infusion of 2,4-dinitrobenzene sulfonic acid (DNBS) and oral administration of dextrane sulfate sodium induced stronger inflammation in CB1-deficient mice (CB1(-/-)) than in wild-type littermates (CB1(+/+)). Treatment of wild-type mice with the specific CB1 antagonist N-(piperidino-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-pyrazole-3-carboxamide (SR141716A) mimicked the phenotype of CB1(-/-) mice, showing an acute requirement of CB1 receptors for protection from inflammation. Consistently, treatment with the cannabinoid receptor agonist R(-)-7-hydroxy-Delta(6)-tetra-hydrocannabinol-dimethylheptyl (HU210) or genetic ablation of the endocannabinoid-degrading enzyme fatty acid amide hydrolase (FAAH) resulted in protection against DNBS-induced colitis. Electrophysiological recordings from circular smooth muscle cells, performed 8 hours after DNBS treatment, revealed spontaneous oscillatory action potentials in CB1(-/-) but not in CB1(+/+) colons, indicating an early CB1-mediated control of inflammation-induced irritation of smooth muscle cells. DNBS treatment increased the percentage of myenteric neurons expressing CB1 receptors, suggesting an enhancement of cannabinoid signaling during colitis. Our results indicate that the endogenous cannabinoid system represents a promising therapeutic target for the treatment of intestinal disease conditions characterized by excessive inflammatory responses.

Figure 1

DNBS- and DSS-induced colitis is worsened in CB1^–/– and in SR141716A-treated (3 mg/kg) C57BL/6N mice. (A and B) Macroscopic score of colonic inflammation (A) and levels of MPO activity (B) in CB1^+/+ and CB1^–/– littermates. (C and D) Macroscopic score of colonic inflammation (C) and levels of MPO activity (D) in vehicle- and SR141716A-treated C57BL/6N mice. Data are means ± SEM.

Figure 2

Histological micrographs showing H&E staining from transverse sections of the colon. (A and B) Colons from CB1^+/+ (A) and CB1^–/– (B) mice without DNBS treatment. (C and D) Colons from CB1^+/+ (C) and CB1^–/– (D) mice 3 days after DNBS treatment. (E and F) Colons from vehicle-treated (E) and SR141716A-treated (3 mg/kg) (F) C57BL/6N mice 3 days after DNBS treatment. In particular, note the severe mucosal infiltration with inflammatory cells, severe submucosal edema, and vascular alterations in CB1^–/– and SR141716A-treated mice. ML, muscular layer; M, mucosa; SM, submucosa. Scale bar: 100 μm.

Figure 3

Pharmacological stimulation of cannabinoid receptors and genetic enhancement of endocannabinoid levels protect against DNBS-induced colitis. (A and B) Macroscopic score of colonic inflammation (A) and levels of MPO activity (B) in vehicle-treated and HU210-treated (0.05 mg/kg) C57BL/6N mice. (C and D) Macroscopic score of colonic inflammation (C) and levels of MPO activity (D) in FAAH^+/+ and FAAH^–/– mice. Data are means ± SEM.

Figure 4

Relative numbers of CB1- and Enk-expressing myenteric neurons are increased 3 days after DNBS treatment, as detected by ISH. (A and B) Micrographs showing CB1 mRNA in untreated (A) and in DNBS-treated (B) CB1^+/+ mice. (C) Quantitative evaluation of CB1-expressing cells in the myenteric plexuses of DNBS-treated CB1^+/+ mice (white bar, n = 3). (D–G) Micrographs showing Enk mRNA in untreated CB1^+/+ (D), untreated CB1^–/– (E), DNBS-treated CB1^+/+ (F), and DNBS-treated CB1^–/– (G) colons. (H) Quantitative evaluation of Enk-expressing cells in the myenteric plexuses of DNBS-treated CB1^+/+ (white bar, n = 3) and CB1^–/– (black bar, n = 3) mice. Values are means ± SEM. *P < 0.05, **P < 0.01 vs. respective untreated controls. Dotted lines, 100%. n = 3 per group. Arrows: CB1- and Enk-expressing cells. Scale bar: 100 μm.

Figure 5

Temporal development of DNBS-induced colitis in CB1^–/– and CB1^+/+ mice. (A) Macroscopic score, (B) MPO activity, and (C) body-weight changes at different time points after induction of colitis. Values are means ± SEM. *P < 0.05.

Figure 6

Intracellular recordings from circular smooth muscles in distal colon of CB1^+/+ and CB1^–/– mice to monitor RMPs. Representative traces are shown for mice before (A), 8 hours after (B), and 24 hours after (C) induction of colitis. In B and C, note the occurrence of oscillatory action potentials in CB1^–/– colons.