Assessment of the role of cannabinoid receptor 2 in innate immune cell trafficking during acute inflammation

Thesis

Activation of the cannabinoid receptor CB₂ has been shown to induce directed leukocyte migration and inhibit leukocyte chemotaxis towards CC chemokines. However, the role that CB₂ plays in regulating macrophage chemotaxis remains understudied. Using a real-time chemotaxis assay and a panel of chemically diverse CB₂ agonists, I set out to examine whether CB₂ modulates primary macrophage chemotaxis. Of 14 agonists tested, only a subset acted as bona fide macrophage chemoattractants. Surprisingly, despite being pertussis toxin-sensitive, neither pharmacological inhibition nor genetic ablation of CB₂ had any effect on CB₂ agonist-induced macrophage chemotaxis. Furthermore, the activation of CB2 had no effect on CCL2 or CCL5- induced macrophage chemotaxis. Therefore, the activation of CB2 does not inhibit CC chemokine-induced macrophage migration and a non-CB₁/CB₂, G_i/o-coupled GPCR must transduce CB2 agonist-induced macrophage chemotaxis. To identify the GPCR responsible, I examined primary murine macrophage GPCR expression and found that they express 124 non-sensory GPCRs. Functional screening of candidate receptors demonstrated that the putative cannabinoid receptors GPR18 and GPR55 and the lipid binding GPCRs LPAR1&5, CYSLTR1&2 and GPER1, were not responsible for CB₂ agonist-induced macrophage chemotaxis. Alongside, a ligand-directed virtual screen, combined with functional testing, uncovered a novel chemotaxis positive chemical scaffold. Importantly, compounds in this series containing a photoaffinity label retained activity and will aid in the identification of the target(s) responsible for CB₂ agonist-induced macrophage chemotaxis in future photocrosslinking experiments. Finally, I assessed whether CB2 controls innate immune cell recruitment in vivo using the zymosan-induced dorsal air pouch inflammation model and animals genetically deleted for CB₂. I found that CB₂^-/- mice had increased air pouch neutrophil and monocyte numbers, as well as pro-inflammatory mediators, during the acute inflammatory phase. Interestingly, mixed bone marrow chimera experiments demonstrated that lack of CB₂ specifically in the myeloid population is responsible for increased neutrophil trafficking. Therefore these data demonstrate that CB₂ acts to regulate neutrophil recruitment during the acute inflammatory response.

Authors: Taylor, L

• Alternative title: Cannabinoid receptor 2 and immune cell trafficking
• Type of award: DPhil
• Level of award: Doctoral
• Awarding institution: University of Oxford
• Language: English
• Keywords: Inflammation; Chemotaxis; Cannabinoids
• Subjects: Immunology; Inflammation