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 (CB), 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 CB in immune regulation might contribute to identify CB 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 CB 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 CB and selectivity over CB. We validate HU210-Alexa488 for the rapid, simultaneous, and reproducible identification of CB in human monocytes, T cells, and B cells by multiplexed flow cytometry. This probe is also suitable for the direct visualization of CB in tonsil tissues, allowing the in vivo identification of tonsil CB-expressing T and B cells. This study provides the first fluorescent chemical tool to investigate CB expression and function in human blood and tonsil immune cells, which might well pave the way to unravel essential features of CB in different immune and ECS-related diseases.

Acute inflammation and pathogenesis of SARS-CoV-2 infection: Cannabidiol as a potential anti-inflammatory treatment?

“…Airway epithelial cells respond to both CB2 receptor-dependent and independent effects of cannabinoids [34]. CB2 is expressed by varieties of immune cells including circulating lymphocytes, monocytes and tissue mast cells and in lymphoid tissues [33,35]. Activation of CB2 receptor can suppress release of inflammatory IL-1, IL-6, IL-12 and TNF-α [36].…”

Highly Selective, Amine-Derived Cannabinoid Receptor 2 Probes

“…In vitro pharmacological assessmento fp repared compounds (vide infra)s howed that amide derivatives 16 and 17 exhibited significantly increased binding affinity to hCB 2 Rc ompared to ester 15,a sw ell as sharplyi ncreased selectivity over hCB 1 R. Interestingly,c annabinoids in which the primary alcohol is replaced by amines and their derivatives have not been studied in detail.T he few examples include an acetamide derivativeo fD 8 -tetrahydrocannabinol (D 8 -THC) reported to be analgesically inactive [33] and more recents tudies of fluorescent probesf or the cannabinoid receptors. [34,35] As mall number of amine analogs was evaluated in mice [36] and baboons, [37] but since these studies predate the discovery and cloning of cannabinoidr eceptors, no data on binding affinities are available.…”

The Role of Cannabinoids in Allergic Diseases: Collegium Internationale Allergologicum (CIA) Update 2020

“…Cannabinoids mainly suppress adaptive T-cell responses by inhibiting proliferation and cytokine production [75,76]. CBD has been shown to induce tolerogenic responses by favouring the generation of regulatory T (Treg) cells [77].…”

“…Their capacity to track CB2 in various cell types and settings may be of quite some interest in studies monitoring the expression of CB2 and its interactions in biological systems (Table 2). Regarding CB1, the conjugation of HU210, a dual agonist of CB1 and CB2, with the fluorescent tag Alexa Fluor 488 resulted in a high-affinity CB1-specific probe [76]. The probe was suitable for use in common biochemical and immunological techniques such as confocal microscopy and flow cytometry.…”

“…The probe was suitable for use in common biochemical and immunological techniques such as confocal microscopy and flow cytometry. It was also validated for the visualization of CB1 in different immune cell subsets [76], suggesting it to be a promising implement for future ECS research in immune-related diseases such as allergy. Besides, novel probes that provide reliable information on the allosteric motifs of CB1 have been designed.…”