Cannabinoid receptor-2 (CB2) agonist ameliorates colitis in IL-10(-/-) mice by attenuating the activation of T cells and promoting their apoptosis

Abstract

Inflammatory bowel disease (IBD) is a chronic intestinal inflammation caused by hyperactivated effector immune cells that produce pro-inflammatory cytokines. Recent studies have shown that the cannabinoid system may play a critical role in mediating protection against intestinal inflammation. However, the effect of cannabinoid receptor induction after chronic colitis progression has not been investigated. Here, we investigate the effect of cannabinoid receptor-2 (CB2) agonist, JWH-133, after chronic colitis in IL-10(-/-) mice. JWH-133 effectively attenuated the overall clinical score, and reversed colitis-associated pathogenesis and decrease in body weight in IL-10(-/-) mice. After JWH-133 treatment, the percentage of CD4(+) T cells, neutrophils, mast cells, natural killer (NK1.1) cells, and activated T cells declined in the intestinal lamina propria (LP) and mesenteric lymph nodes (MLN) of mice with chronic colitis. JWH-133 was also effective in ameliorating dextran sodium sulfate (DSS)-induced colitis. In this model, JWH-133 reduced the number and percentage of macrophages and IFN-γ expressing cells that were induced during colitis progression. Treatment with aminoalkylindole 6-iodo-pravadoline (AM630), a CB2 receptor antagonist, reversed the colitis protection provided by JWH-133 treatment. Also, activated T cells were found to undergo apoptosis following JWH-133 treatment both in-vivo and in-vitro. These findings suggest that JWH-133 mediates its effect through CB2 receptors, and ameliorates chronic colitis by inducing apoptosis in activated T cells, reducing the numbers of activated T cells, and suppressing induction of mast cells, NK cells, and neutrophils at sites of inflammation in the LP. These results support the idea that the CB2 receptor agonists may serve as a therapeutic modality against IBD.

Published by Elsevier Inc.

Figures

Fig. 1. Change in body weight, histological characterization and inflammation score after JWH-133 treatment in C57BL/6 and IL-10−/− mice

IL-10−/− mice, on a C57BL/6 background, and normal C57BL/6 mice (▵) received, 1, 2.5 and 5 mg/kg body weight of JWH-133, (◻), (○), (∎) or vehicle (●), every second day, starting at the beginning of week 18 when mice had lost about 15-17 % of their body weight and continuing until the week 25. The mice were sacrificed two weeks after the last injection at week 27. The body weight of the IL-10−/− mice was recorded every week, and the change from initial body weight was expressed as a percentage change in body weight (Fig. 1A). Histological sections of colons from the pre-colitis and two groups of IL-10−/− mice at the week 27 were presented (as described in Fig 1C). Vehicle treated mice (Fig. 1 C lower panel) showed significant lymphocyte infiltration and distortion of glands, while JWH-133-treated mice (Fig. 1 B upper panel) showed markedly decreased lymphocyte infiltration. Other pathologic changes included diffuse leukocyte infiltrates, distorted crypts, and thickening of the lamina propria in the area of distorted crypts in the colon. The histological inflammation scores of these two groups are presented in (Fig. 1 C). Asterisks (*) indicate statistically significant differences (p < 0.01) between vehicle and JWH-133 treated groups. Representative sections from three separate experiments (10 and 40X magnification) are shown, with each group containing six mice.

Fig. 2. Effect of JWH-133 on T cells during colitis

Splenic, MLN and LP lymphocytes were isolated from pre-colitis mice and two groups of IL-10−/− mice (at 27 weeks) after the end-points of experiment as described in Fig 1 legend. The lymphocytes were stained for CD4+ and CD8+ T cells and analyzed using flow cytometry. The numbers in the bottom right quadrant indicate the total percentage of CD4+ T cells (Panel A); the upper left quadrant indicates the total percentage of CD8+ T (Panel A) cells. The absolute number (mean of 6 samples ± SEM) of CD4+ T cells from spleen, MLN and LP in each group was enumerated and depicted (Panel B). We did not notice any major changes in the CD8+ T cells in any experimental group. Asterisks (*) indicate statistically significant difference (p < 0.01) between vehicle vs JWH-133.

Fig. 3. JWH-133 reduces percentage and numbers neutrophil during colitis

Spleen, MLN and LP lymphocytes were isolated from pre-colitis IL-10−/− mice and at the week 27 from IL-10−/− mice that received 2.5 (mg/kg body weight) of JWH-133 and vehicle control. The lymphocytes were stained for CD3+ T and neutrophil (LY6G+) cells. The spleen, MLN or LP-derived CD3− lymphocytes were characterized for neutrophil expression by flow cytometry. The numbers in the upper left quadrant indicate the total percentage of neutrophil. Data represent the total number of cells ± SEM from three independent experiments involving 6 mice per groups. Asterisks (*) indicate statistically significant differences (p < 0.01) between vehicle and JWH-133 treated groups.

Fig. 4. JWH-133 reduces the induction of activated T cells during colitis

Spleen, MLNs and LP lymphocytes were isolated from the pre-colitis and two groups of IL-10−/− mice at the week 27 and stained for CD4+ and CD69+ T cells. Panel A shows a representative experiment indicating the percentages of various types of cells and panel B depicts the absolute numbers of these cells in various lymphoid organs. In panel B, the data represent the total number of cells ± SEM from three independent experiments. Asterisks (*) indicate statistically significant differences (p < 0.01) between vehicle and JWH-133 treated groups.

Fig. 5. JWH-133 treatment mediates mast and NK1.1 during colitis

Spleen and LP cells were isolated from IL-10−/− mice before the onset of colitis (pre-colitis) and two groups of IL-10−/− mice post-colitis at the week 27. The cells were stained for expression of CD3 and NK1.1 or CD117 (mast cell). Panel A and B show a representative experiment indicating the percentages and numbers of NK1.1 cells in various lymphoid organs. Panels C, and D represent the total number and percentage of mast cells ± SEM from three independent experiments. Asterisks (*) indicate statistically significant differences (p < 0.01) between vehicle and JWH-133 treated groups.

Fig. 6. JWH-133 treatment induces apoptosis both in-vitro and in-vivo in the colon and inhibits T cell proliferation

Lymphocytes obtained from MLN (~ 75% T cells) of normal C57BL/6 mice were cultured after anti-CD3/CD28 Abs activation in the presence of vehicle or JWH-133 (0, 1, 5, 10, 20 and 50 μM). Some cultures received JWH+ 20 or 50μM of AM630. The apoptosis of T cells was measured by flow cytometry (A). In situ tunnel assay sections of colons from the pre-colitis and two groups of IL-10−/− at the week 27 (as described in Fig 1) and normal C57BL/6 mice (8 weeks) were presented (B). Representative sections from three separate experiments (20X magnification) are shown. Vehicle treated normal as well as well as IL-10−/− mice showed less apoptosis as compared to JWH-133-treated mice. The percentage of apoptosis scores for each group is presented (C). Panel D shows BrdU cell proliferation assay performed using lymphocytes obtained from MLN (~ 75% T cells) of normal C57BL/6 mice cultured with anti-CD3/CD28 Abs in the presence of vehicle or JWH-133 (0, 5 and 50 μM) at a density of 5 × 106 cells/ml. The Proliferation of T cells were measured by BrdU incorporation. The panel D data presented are the mean OD450 for proliferation ± SEM of quadruplicate cultures. The statistical significance between experimental and control group was assessed using Student’s t tests. Data represent the mean of three independent experiments involving six mice per group. Asterisks (*) indicate statistically significant differences (p < 0.01) between groups.

Fig. 7. Change in body weight, colitis score and severity of DSS-induced colitis after JWH-133 and AM-630 treatment

BL/6 mice were given vehicle (▵, control), JWH-133 alone (20 mg/kg body weight)(◻), DSS alone (3%) in drinking water (∎, DSS), or a combination of DSS with 10 (▴) or 20 (●) mg/kg body weight dose of JWH-133 or a combination of JWH-133 (20 mg/kg) + 20 mg/kg dose of AM-630 (○) for fourteen days. DSS was replaced after seven days with a water cycle (ad libitum) for another seven days. The body weight of these mice was recorded daily. Changes in body weight were expressed as a percentage of the weight (panel A). Panel B represents the persistence or improvement of colitis inflammation scores. Histological sections of colons from the 4 groups of mice were presented (panel C). DSS induced mice showed significant lymphocyte infiltration and distortion of glands (arrow), while JWH-133 treated mice showed colon lumen having markedly decreased lymphocyte infiltration. The AM-630 treatment further increased leukocyte infiltrates, distorted crypts (arrow) in the colon. The statistical significance between values of each group was assessed by Student’s t test. Data represents the mean of three experiment involving 6 mice per group.

Fig. 8. Effect of JWH-133 treatment on CD11b+ and IFN-γ+ expression during colitis

Colitis was induced using DSS and these mice were treated with JWH-133 and AM630 as described in Fig 7 legend. Cells from the spleen, MLN and LP were stained for CD11b+ (panel A) macrophages and for IFN-γ+ (panel B) cells using flow cytomtery.