Gestational exposure to the cannabinoid WIN 55,212-2 and its effect on the innate intestinal immune response

Abstract

The consequences of marijuana consumption during pregnancy and its effects on the function of the immune system have been little studied. Marijuana is one of the most consumed recreational drugs among pregnant women, and it is known that gestational exposure to marijuana can have serious effects on the offspring after birth. In this study, we challenged the immune system of Wistar rats by infecting them with the parasitic nematode Trichinella spiralis. A treatment group of these animals was prenatally exposed to the cannabinoid WIN 55,212-2; a control group was not exposed. At 5 days of infection, the treated animals were less effective in eliminating intestinal parasites; moreover, this effect was correlated with a deficiency in mucus production, lower recruitment of eosinophils in the duodenum, and a reduced percentage of Tγδ and NK cells. In conclusion, the gestational administration of the synthetic cannabinoid WIN 55,212-2 induces lasting changes to the function of the immune system against infection with T. spiralis in male Wistar rats, making them more susceptible to infection.

Figure 1

(A) Structure of the main cannabinoid of the Cannabis sativa plant, tetrahydrocannabinol, THC (I). General structure of aminoalkylindole compounds (II), derived from pravadoline, which have the same biological effects as THC, with WIN 55,212-2 being the most representative compound (III). (B) Parasitic load in the duodenum of the three experimental groups. Animals were infected with 2000 muscular larvae by intragastric route and sacrificed after 5 days post-infection. The larvae were recovered from the gut of infected animals. Data are expressed as mean ± SD. One-way ANOVA test and post hoc Tukey multiple comparisons test was performed. *p < 0.05; n = 6 for each group.

Figure 2

Morphology of duodenum. Hematoxylin and eosin staining (H&E; 4×) in non-infected (a,b,c) and infected (d,e,f) animals. IG: intestinal glands; ML: mucosal layer; S: serosa; SL: submucosal layer. Black arrows show damage at intestinal-gland level.

Figure 3

Morphology of glands in the duodenum. (A) Representative gland images from control (a,d), vehicle (b,e) and WIN 55,212-2 (c,f) experimental groups, in either non-infected (a,b,c) or infected (d,e,f) groups. Black arrows show goblet cells. The square (g) shows a representative eosinophil with ematoxylin and eosin stain (H&E; 40X). (B) Quantification of eosinophils. Data are expressed as mean ± SD. Two-way ANOVA test and post hoc Tukey multiple comparisons test were performed. Differences among treatments are shown with bars. Letters above each column indicate statistical differences among groups due to infection: a, no significant difference compared with a; b, no significant difference compared with b; p < 0.05 for a compared to b, or vice versa; n = 6 for each group.

Figure 4

Morphology of villi in the duodenum. (A) Representative villi images from control (a,d), vehicle (b,e) and WIN 55,212-2 (c,f) experimental groups, in either non-infected (a,b,c) or infected (d,e,f) groups. Black arrows show goblet cells. The square (g) shows a representative eosinophil with hematoxylin and eosin stain (H&E; 40X). (B) Eosinophil quantification. Data are expressed as mean ± SD. Two-way ANOVA test and post hoc Tukey multiple comparisons test were performed. Differences among treatments are shown with bars. Letters above each column indicate statistical differences among groups due to infection: a, no significant difference compared with a; b, no significant difference compared with b; p < 0.05 for a compared to b, or vice versa; n = 6 for each group.

Figure 5

Goblet cells in glands of the duodenum. (A) Representative goblet cell images in the glands of the duodenum in control (a,d), vehicle (b,e) and WIN 55,212-2 (c,f) experimental groups, in either non-infected (a,b,c) or infected (d,e,f) groups. Goblet cells are stained blue. (B) Goblet cell quantification according to filling degree (e: empty, he: half-empty, f: full), with Alcian blue staining (40X). Data are expressed as mean percentage ± SD; n = 6 for each group.

Figure 6

Goblet cells in villi of the duodenum. (A) Representative goblet cell images in the villi of the duodenum in control (a,d), vehicle (b,e) and WIN 55,212-2 (c, f) experimental groups, in either non-infected (a,b,c) or infected (d,e,f) groups. Goblet cells are stained blue. (B) Goblet cell quantification according to filling degree (e: empty, he: half-empty, f: full), with Alcian blue staining (40X). Data are expressed as mean percentage ± SD; n = 6 for each group.

Figure 7

Percentage of NK and Tγδ cells in duodenum. (A) Representative dot plots in control, vehicle and WIN 55,512-2 groups of non-infected and infected groups. (B) Percentage of Tγδ and NK cells. Data are expressed as mean ± SD. Two-way ANOVA test and post hoc Tukey multiple comparisons test were performed. **p < 0.01; differences among treatments are shown with bars. Letters above each column indicate statistical differences among groups due to infection: a, no significant difference compared with a; b, no significant difference compared with b; p < 0.05 for a compared to b, or vice versa; n = 6 for each group.

Figure 8

Summary of changes in the duodenum of adult rats prenatally exposed to WIN 55,212-2. Pregnant rats were treated with the synthetic cannabinoid WIN 55,212-2, and adult offspring were used for the posterior studies (A). The morphology of the duodenum and the percentage of Tγδ and NK cells were analyzed in the non-infected (B) and infected (C) adult offspring with Trichinella spiralis. Arrows indicate the increased or decreased values of the evaluated parameters, whereas “X” indicates their absence.