Cannabinoids are bioactive signaling molecules exerting several effects, including modulation of immune function. One of the agents studied for its immunosuppressive activity, Δ9-tetrahydrocannabinol (Δ9-THC), is thought to exert part of its activity on immune cells via interaction with cannabinoid receptors 1 (CB1) and 2 (CB2). In previous studies Δ9-THC caused an increase in steady state viral hemagglutinin RNA levels, suggesting decreased viral clearance due to compromised immune function. In addition, in CB1 and CB2 double knock out (CB1-/-CB2-/-) mice, a reduced viral burden and greater magnitude in the immune response were observed, suggesting a role for CB1 and/or CB2 in regulating immune function. The identity of the functionally dysregulated immune population(s) as a result of CB1 and CB2 deletion were unknown. In addition, the effect of Δ9-THC treatment on the cellular immune response to influenza remained to be elucidated. Overall, this dissertation project was designed to test the hypothesis that Δ9-THC suppresses immune responses generated by influenza virus by perturbing an endogenous signaling pathway relying in part on the presence of CB1 and/or CB2. In the present studies, Δ9-THC suppressed cytotoxic T lymphocyte (CTL) function independent of CB1 and CB2, suggesting an alternate mechanism for immune modulation by Δ9-THC in these cells. Second, a kinetic study of influenza infection demonstrated greater immune response magnitude in CB1-/-CB2-/- compared to WT mice. This was evidenced by elevated steady-state mRNA levels for genes involved in the inflammatory response, earlier T cell activation, greater percentages of cytokine-secreting effector cells, and increased immunopathology in lung tissue. These events likely lead to lower viral burden at the expense of greater tissue damage in CB1-/-CB2-/- mice compared to WT. These differences observed in vivo were not reproduced by stimulation of T cells in vitro, suggesting differences in antigen presenting cells (APC), which elicit effector cells. Dendritic cells (DC) generated from bone marrow (bmDC) and alveolar macrophages (AM) isolated from CB1-/-CB2-/- mice had greater maturation with and without Toll-like receptor (TLR) stimulation compared to WT mice and bmDC elicited antigen-specific T cells without the need for a maturation stimulus. Third, focusing on the peak day of the inflammatory response induced by influenza virus at day 3, the immune response of APC was characterized in WT and CB1-/-CB2-/- mice in the presence of Δ9-THC. Δ9-THC suppressed influx of APC, including conventional DC (cDC), plasmacytoid DC (pDC), and AM into the lung after influenza infection in WT only. While there was no change in maturation of lung-isolated APC due to Δ9-THC treatment, only mature APC reach the lung, thus Δ9-THC might have affected maturation of APC prior to migration to the lung. Indeed, Δ9-THC suppressed DC maturation and ability to elicit antigen-specific T cells in vitro. Taken together, Δ9-THC impairs the immune response to influenza, in part, by suppressing APC function in a CB1 and/or CB2 dependent manner, while suppressing CTL elicitation in a CB1 and CB2 independent manner. Also, the presence of CB1 and/or CB2 on APC prevents exacerbated immune responses after influenza infection. These studies suggest that CB1 and/or CB2 maintain immune homeostasis and prevent excessive reactivity after immune stimulation.
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