A Fluorescent Probe to Unravel Functional Features of Cannabinoid Receptor CB₁ in Human Blood and Tonsil Immune System Cells

Abstract

The human endogenous cannabinoid system (ECS) regulates key physiological processes and alterations in its signaling pathways, and endocannabinoid levels are associated with diseases such as neurological and neuropsychiatric conditions, cancer, pain and inflammation, obesity, and metabolic and different immune related disorders. Immune system cells express the G-protein coupled cannabinoid receptor 1 (CB₁), but its functional role has not been fully understood, likely due to the lack of appropriate tools. The availability of novel tools to investigate the role of CB₁ in immune regulation might contribute to identify CB₁ as a potential novel therapeutic target or biomarker for many diseases. Herein, we report the development and validation of the first fluorescent small molecule probe to directly visualize and quantify CB₁ in blood and tonsil immune cells by flow cytometry and confocal microscopy. We coupled the cannabinoid agonist HU210 to the fluorescent tag Alexa Fluor 488, generating a fluorescent probe with high affinity for CB₁ and selectivity over CB₂. We validate HU210-Alexa488 for the rapid, simultaneous, and reproducible identification of CB₁ in human monocytes, T cells, and B cells by multiplexed flow cytometry. This probe is also suitable for the direct visualization of CB₁ in tonsil tissues, allowing the in vivo identification of tonsil CB₁-expressing T and B cells. This study provides the first fluorescent chemical tool to investigate CB₁ expression and function in human blood and tonsil immune cells, which might well pave the way to unravel essential features of CB₁ in different immune and ECS-related diseases.