EXPOSURE OF ADOLESCENT MICE TO Δ9-TETRAHYDROCANNABINOL SHAPES IMMUNE RESPONSE IN ADULTHOOD

Marijuana, also called Cannabis, is the illicit drug most frequently used by human adolescents;this is facilitated by the fact that users generally perceive these drugs as relatively harmless. Recent data estimate that cannabis use starts in the period from 12 to 18 years of age, though even earlier use (9\u201310 years) is now anecdotally reported. Among the many consequences related to the cannabis use, it is increasingly emerging as the \u3949-THC, as well as endogenous cannabinoids, are able to affect certain functions of the immune system. In fact, studies conducted in experimental animals indicate that cannabis impairs the immune response by altering the homeostasis of the immune system. The adolescence is a particularly vulnerable period of individual life, both for physical and psychological maturity; also the immune system during this development period might be particularly vulnerable to the effects induced by this drug. However, there aren't currently available studies to investigate whether the cannabis intake during adolescence period can have a negative impact on some immune functions such as the individual's response to infection, allergic and autoimmune pathologies, both during the period of drug intake and in adulthood, thus long time from the last direct cannabis exposure. The purpose of this study was to investigate whether the use of cannabis during adolescence may induce immediate and delayed effects on the immune system that may then persist in adulthood. As immune parameters we considered both innate and acquired immunity, measuring T-cells and macrophage cytokines production. We used Balb C/J male mice, 33 post natal days (PND) as model of adolescence age and 80PND as model of adult age. \u3949-THC was administerd with increasing subcutaneous (s.c.) doses, starting from 5 mg/kg and gradually reaching 15mg/kg in 10 days. Control animals were treated with the vehicle for the same duration of time. Some groups of animals were sacrificed, for the immune evaluation, immediately at the end of the treatment, while other groups of mice were housed until 90 PND before the immune evaluation. We recently added a fourth study group consisting of adult animals treated with the drug, for 10days, and housed for 47 days, up to 137 PND, when the evaluation of immune parameters were performed. The data regarding this last study group are not completed, due to the long times required for the implementation of the experimental protocol and refer only to the innate and acquired immunity. In order to study acquired immunity, young and adult mice were immunized with KLH protein to induce an antigen-specific reaction and Th1/Th2 balance was assessed by measuring the production of IFN-\u3b3, IL-4 and IL-10 by splenic lymphocytes. In order to assess macrophage function we used peritoneal macrophages stimulated in vitro with LPS endotoxin; IL-1\u3b2 and TNF-\u3b1 were assessed as pro-inflammatory cytokines, while IL-10 was evaluated as anti-inflammatory cytokine. The cytokine production was measured by specific ELISA and Real-Time PCR. At the end of \u3949-THC treatment in both young and adult mice, after immunization, a significant reduction in the production of IFN-\u3b3 was observed, while the Th2 cytokines IL-4 and IL-10 were significantly increased. When we measured immune response of adult mice treated with the drug in adolescence, we observed that the production of IFN-\u3b3 was still lower and that, in contrast to what observed immediately at the end of treatment, also the Th2 cytokine levels were decreased compared with vehicle mice. In adult animals, assessed 47 days after the end of treatment, there were no significant differences between \u3949-THC or vehicle groups. We also evaluated the ability of the animals to develop an antibody response by measuring IgM anti-KLH titers in the serum; in all study groups, the antibody titers were significantly reduced compared to the vehicle. Regarding the macrophage function, the \u3949-THC treatment induced in both young and adult mice a significant increase of IL-10 while TNF-\u3b1 and IL-1\u3b2 are decreased. Particularly interesting were the results obtained in mice treated in adolescent age with \u3949-THC respect to vehicle; in fact, we observed an opposite effect since IL-1\u3b2 and TNF-\u3b1 levels were significantly increased while IL-10 production was lower, indicating a switch towards a pro-inflammatory phenotype of the macrophage. These effects on cytokines are at transcriptional level since we observed similar result by measuring IL-10, IL-1\u3b2 and TNF-\u3b1 mRNA with RT-PCR. In adult animals evaluated 47 days after the end of treatment, by analyzing the pro- and anti-inflammatory cytokine levels and their relative mRNA expression, no significant differences were found between treatments considered. In order to understand whether the \u3949-THC effects might be due to an alteration of circulating hormones, we also assessed the modulation of hypothalamic pituitary-adrenal axis (HPA) axis activity, after sub-acute and chronic \u3949-THC treatment, by measuring the corticosterone plasma levels in the three study groups. The corticosterone concentrations were lower in all groups, indicating a long lasting dysregulation of HPA. Using a method HPLC/ Mass Spectrometry, we also evaluated blood levels of \u3949-THC and its main metabolites in the experimental groups considered, showing that at the end of 10 days treatment they were comparable to those that can be found in human heavy smokers. We also demonstrated that in animals treated as adolescents and studied as adults, plasma \u3949-THC was not more present, further assuring that the effects observed were due to a long lasting modulation of immunity In addition, to understand the mechanisms underlying the observed effects, we began to investigate whether the \u3949-THC treatment may affect hematopoietic cells development. Preliminary data obtained by evaluating the number of colonies formed by hematopoietic precursors have not highlighted differences between the different treatment groups. Finally, we started to study also the \u3949-THC effects on neuroinflammation, by evaluating the basal production of pro- and anti-inflammatory cytokines in brain areas that are particularly rich in cannabinoid receptors, such as hippocampus and hypothalamus. Our data indicate that chronic treatment with the drug is able to reduce the basal levels of IL-1\u3b2 and TNF-\u3b1 in the brain areas considered, in adolescent and adult mice assessed immediately at the end of treatment. The basal production of IL-10 instead appears to be increased compared to relative vehicles in mice evaluated immediately at the end of the treatment. In adult animals, treated with the drug as adolescents, the basal production of pro-inflammatory cytokines, in hypothalamus and in hippocampus, appears to be increased with respect to vehicles and IL-10 decreased, indicating a predisposition to a pro-inflammatory status. Although these data are preliminary they indicate a parallel modulation of brain and peripheral cytokines by \u3949-THC. In conclusion, these results indicate that the immune system is profoundly altered by treatments with \u3949-THC, with the presence of a dysregulated response. In particular we can affirm that the administration of \u3949-THC in adolescent animals has significant effects on the immune response that last long after its intake