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A fluorescent probe to unravel functional features of cannabinoid receptor CB1 in human blood and tonsil immune system cells Mar Martín-Fontecha, Alba Angelina, Beate Rückert, Ainoa Rueda- Zubiaurre, Leticia Martin-Cruz, Willem van de Veen, Mübeccel Akdis, Silvia OrtegaGutierrez, María L. López-Rodríguez, Cezmi Akdis, and Oscar Palomares Bioconjugate Chem., Just Accepted Manuscript • DOI: 10.1021/acs.bioconjchem.7b00680 • Publication Date (Web): 09 Jan 2018 Downloaded from http://pubs.acs.org on January 10, 2018

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Bioconjugate Chemistry

Martín-Fontecha et al. 1

A fluorescent probe to unravel functional features of cannabinoid receptor CB1 in

2

human blood and tonsil immune system cells

3 4

Mar Martín-Fontecha,a Alba Angelina,b Beate Rückert,c Ainoa Rueda-Zubiaurre,a

5

Leticia Martín-Cruz,b Willem van de Veen,c Mübeccel Akdis,c Silvia Ortega-Gutiérrez,a

6

María Luz López-Rodríguez,a Cezmi A. Akdis,c Oscar Palomaresb*

7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23

a

Department of Organic Chemistry, School of Chemistry, Complutense University of Madrid, Madrid, Spain b Department of Biochemistry and Molecular Biology, School of Chemistry, Complutense University, Madrid, Spain c Swiss Institute of Allergy and Asthma Research (SIAF), University of Zurich, Davos, Switzerland

*Corresponding Author:

Oscar Palomares, PhD Department of Biochemistry and Molecular Biology, Chemistry School, Complutense University of Madrid, Ciudad Universitaria s/n, 28040 Madrid, Spain. Telephone: + 34 913944161 Fax: + 34 913944159 Email: [email protected]

1 ACS Paragon Plus Environment

Bioconjugate Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Abstract

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The human endogenous cannabinoid system (ECS) regulates key physiological

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processes and alterations in its signalling pathways and endocannabinoid levels are

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associated to diseases such as neurological and neuropsychiatric conditions, cancer, pain

28

and inflammation, obesity, metabolic and different immune related disorders. Immune

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system cells express the G-protein coupled cannabinoid receptor 1 (CB1) but its

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functional role has not been fully understood, likely due to the lack of appropriate tools.

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The availability of novel tools to investigate the role of CB1 in immune regulation might

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contribute to identify CB1 as a potential novel therapeutic target or biomarker for many

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diseases. Herein, we report the development and validation of the first fluorescent small

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molecule probe to directly visualize and quantify CB1 in blood and tonsil immune cells

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by flow cytometry and confocal microscopy. We coupled the cannabinoid agonist

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HU210 to the fluorescent tag Alexa Fluor 488, generating a fluorescent probe with high

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affinity for CB1 and selectivity over CB2. We validate HU210-Alexa488 for the rapid,

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simultaneous, and reproducible identification of CB1 in human monocytes, T and B

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cells by multiplexed flow cytometry. This probe is also suitable for the direct

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visualization of CB1 in tonsil tissues, allowing the in vivo identification of tonsil CB1-

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expressing T and B cells. This study provides the first fluorescent chemical tool to

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investigate CB1 expression and function in human blood and tonsil immune cells, which

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might well pave the way to unravel essential features of CB1 in different immune and

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ECS-related diseases.


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GRAPHICAL TABLE OF CONTENTS

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CB1 visualization in tonsils

Lymphocytes 59.4%

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CB1 quantification in blood

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The human endogenous cannabinoid system (ECS) is a complex signalling network

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involved in a large number of physiological processes.1,

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endocannabinoid ligands, the proteins related to their synthesis and degradation and the

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cannabinoid receptors (CBRs).2 The endocannabinoids anandamide (AEA) and 2-

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arachidonoylglycerol (2-AG) bind to the G-protein coupled cannabinoid receptor 1

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(CB1) and 2 (CB2). CB1 is largely expressed in the central nervous system and also in

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peripheral tissues and immune cells.2-4 The high-resolution crystal structure of human

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CB1 and agonist-bound complexes have been recently reported.5-7 CB2 is mainly

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expressed in immune cells and also in other cell types.2,

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triggers a complex network of signalling pathways leading to the regulation of key

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physiological processes such as proliferation, differentiation, and cell survival.2-4,

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Alterations in the ECS signalling pathways and changes in the endocannabinoid levels

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have been associated to different diseases such as neurological and neuropsychiatric

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conditions, cancer, pain, inflammation, obesity, metabolic disorders, septic shock and

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different immune system related disorders.2, 8-11

2

3

The ECS comprises the

The activation of CBRs

8

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The immune system encompasses a complex interactive network of cells and

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molecules that protect the host against potentially dangerous pathogens, while keeping a

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state of tolerance to self and innocuous non-self antigens.12,

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system employs a plethora of tightly regulated mechanisms, and alterations in these

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processes lead to immune tolerance-related diseases such as autoimmunity, tumour

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tolerance, rejection of organ transplants or allergy. For long time, it was thought that

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CB1 and CB2 modulated neurological and immune functions, respectively. However,

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several studies demonstrated that immune system cells also express functional CB1 that

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could play a role in the immune regulation of T cells, B cells and innate immune cells

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such as monocytes, dendritic cells (DCs) or macrophages.8, 14-18 Therefore, there is an


13

For that, the immune

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increased interest in studying the role of CB1 in the control of immune responses. The

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better understanding of the role of CB1 in immune regulation might well contribute to

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identify CB1 as a potential novel therapeutic target as well as to determine whether it

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can be a candidate biomarker of disease, prognosis or treatment response.

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In the particular case of allergic diseases, mouse models showed different roles for

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the ECS.19-23 In humans, CB1 plays a potent inhibitory role on mast cell activation in the

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airway mucosa and skin.24, 25 We showed that the mRNA expression levels of CB1 are

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upregulated in tonsils and peripheral blood immune cells of allergic patients.16 Although

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it is plausible that the ECS may contribute to the regulation of allergic diseases, the

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underlying molecular mechanisms are not fully understood and human data are still

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scarce. In the same way, whether CB1 might represent a potential novel biomarker in the

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context of allergy or other immune tolerance-related diseases remains completely

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unknown and requires further investigations. Up to date, the lack of appropriate tools

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has hampered these particular studies. In this regard, antibodies for CB1 could be

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employed, but they have important limitations in terms of sensitivity and specificity

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likely due to the poor immunogenicity reported for CBRs.26, 27 In addition, the batch-to-

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batch variations inherent the way in which antibodies are produced has been recognized

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as an important problem of accuracy and reliability.28, 29 Therefore, there is a need for

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the development of tools that enable the direct visualization and quantification of CB1 in

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immune cells. The availability of such tools would allow the quantification of up- or

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down-regulation of CB1 as a disease biomarker in a straightforward manner both in

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peripheral blood and tissue samples, which might well contribute to stratify patients for

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personalized medicine.

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In this work, we develop and validate the first fluorescent small molecule probe that

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allows the visualization and quantification of CB1 in peripheral blood and tonsil immune




cells. For this purpose, we coupled the synthetic cannabinoid agonist HU21030 to the

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fluorescent tag Alexa Fluor 488. The resulting fluorescence probe displays high affinity

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for CB1 and selectivity over CB2. We validate this probe as a chemical tool for the

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rapid, simultaneous and reproducible identification of functional CB1 in human

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monocytes, T cells and B cells from peripheral blood and tonsils by flow cytometry in a

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single multiplexed manner. In addition, we demonstrate that HU210-Alexa488 is

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suitable for the visualization of CB1 in tonsil tissues, which allows the ex vivo

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identification of tonsil T and B cell subsets expressing this receptor. This study provides

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the first fluorescent chemical tool to interrogate CB1 expression in blood and tonsil

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immune cells, which might well pave the way to unravel the functional features of CB1

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and to uncover the potential role of this receptor as a novel biomarker not only for

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allergy but also for other immune related diseases. The applicability of this validated

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fluorescent probe could be also extended to other ECS-related diseases.


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RESULTS AND DISCUSSION

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Development of a CB1-specific fluorescence small molecule probe

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A small molecule fluorescent probe to visualize and quantify CB1 should contain three

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main components: i) a high affinity CB1 ligand with a position amenable for

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derivatization, ii) an appropriate fluorophore and iii) a suitable spacer to avoid potential

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steric interferences of the bulky fluorophore that could produce the loss of affinity for

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the receptor under study (Figure 1A). Regarding the ligand, among the cannabinoids

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that could be initially considered, the main endocannabinoids (AEA, 2-AG) were ruled

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out because our previous studies revealed that the attachment of tags to these ligands

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reduces the affinity of the probes, which restricts their use to the visualization of the

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CBRs only to transfected cells.31 We focused our efforts on the synthetic high-affinity

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cannabinoid ligand HU210 [Ki (CB1) = 0.061 nM, Ki (CB2) = 0.52 nM],30 because we

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previously demonstrated that this ligand admits the introduction of biotin in the free

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allylic hydroxyl group without any important loss of affinity (Figure 1B).16,

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Biotinylated probes 1 and 2 have been successfully used for the visualization of CB1 in

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neurons and in different immune cells, but they require a two-step labelling process and

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additional steps to block endogenous biotin, which do not make them suitable for flow

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cytometry clinical routine or tissue staining. To avoid these drawbacks, we selected the

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bright fluorescent Alexa Fluor 488 dye that is commonly used for flow cytometry and

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confocal microscopy imaging. The spacer between the ligand and fluorophore consists

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of a 6-aminocarbonyl linker that will be attached to the HU210 through amide

133

functionality to avoid the potential lability of the ester group (Figure 1A).

134


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Martín-Fontecha et al. A

B

135 136

Figure 1. Development of a CB1 fluorescent probe. (A) Scheme of the main

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components of a fluorescent probe and structure of the HU210-Alexa Fluor 488 probe

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3. (B) Structure of the cannabinoid ligand HU210 and biotin-based probes 1 and 2

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previously described.

140 141

The preparation of the desired fluorescent compound 3 involved the synthesis of the

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HU210 scaffold functionalized as the corresponding allylic amine, followed by the

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incorporation of the appropriate spacer and further coupling with the commercially

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available tetrafluorophenyl (TFP) ester of Alexa Fluor 488 (Scheme 1). Thus, phenol

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433 was protected as triisopropylsilyl (TIPS) ether under microwave (MW) irradiation,

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followed by removal of the pivaloyl group with lithium aluminum hydride to afford

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alcohol 5, which was transformed into the corresponding allylic amine 7 through

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Mitsunobu reaction with phthalimide and subsequent deprotection, using hydrazine as

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the cleaving reagent. Condensation of 7 with 6-(N-Boc-amino)hexanoic acid and further

150

removal of TIPS and Boc protecting groups yielded 6-aminohexanamide 10, which was

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finally coupled with Alexa Fluor 488 TFP ester to afford fluorescent derivative 3.


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153 154

Scheme 1 Synthesis of the fluorescent probe 3. Reagents and conditions: (a) TIPS-Cl,

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imidazole, DMF, MW, 200 ⁰C, 95%; (b) LiAlH4, THF, 0 ⁰C, 72%; (c) phthalimide, PPh3,

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DEAD, THF, rt, 93%; (d) i) N2H4·H2O, EtOH, reflux; ii) HCl/H2O 1:1 reflux to rt, 96%; (e) 6-

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(N-Boc-amino)hexanoic acid, DCC, DCM, rt, 74%; (f) TBAF, THF, 0 ⁰C, 81%; (g) TFA, DCM,

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rt, 84%; (h) Alexa Fluor 488 TFP ester, DCM, DMF, rt, 66%.

159 160

Affinity of synthesized compound 3 for CB1 and CB2 was assessed by radioligand

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competitive binding assays using membranes of HEK-293-EBNA cells transfected with

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human CB1 and CB2, respectively, and [3H]-CP55940 as radioligand. Interestingly,

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fluorescent probe 3 exhibited affinity values in the nanomolar range toward CB1, while

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showing almost no affinity for CB2 (submicromolar range) [Ki (CB1)=27±4 nM; Ki

165

(CB2)=0.8±0.2 μM]. Therefore, the newly identified fluorescent derivative 3 displays an

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optimized proﬁle in terms of aﬃnity and selectivity (30-fold over CB2) to carry out its

167

validation as a chemical tool to interrogate CB1 in immune system cells.

168




Validation of HU210-Alexa488 probe 3 to detect and quantify CB1-expressing

170

immune cells by flow cytometry

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To assess the capacity of the new fluorescent probe 3 to identify and quantify peripheral

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blood CB1-expressing immune cells by flow cytometry, we isolated peripheral blood

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mononuclear cells (PBMC) from healthy donors and stained with probe 3 (1 μM) or

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with Alexa Fluor 488 alkyne (1 μM) as control (Figure 1A). We analysed the expression

175

of CB1 in lymphocytes and monocytes by gating according to cell size and granularity

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(Figure 2A). Around 60% of lymphocytes and 80% of monocytes stained positive with

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HU210-Alexa488 fluorescent probe 3, whereas only around 1% and 4%, respectively,

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did so with control Alexa Fluor 488 alkyne (Figure 2A). As negative control, we also

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used the HEK293T cell line (Figure 2B), which does not constitutively express CBRs.

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HEK cells did not stain positive with probe 3 beyond the basal background detected

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with the control supporting that the probe 3 did not show non-specific binding (Figure

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2B). An excess of unlabelled HU210 (50 μM), but not of the CB2 selective agonist

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HU3084, 30 (50 μM) significantly shifted the positive staining detected with probe 3 (1

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μM) in both lymphocytes and monocytes (Figure S1), thus confirming the specificity of

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this fluorescent probe to detect CB1-expressing cells in peripheral blood by flow

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cytometry. These data strongly suggest that the fluorescent signal provided by probe 3

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is due to CB1 but not to CB2 expression. We also analysed the stained PBMC and HEK

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cells after cytospin by confocal microscopy (Figure 2C). CB1-expessing cells were only

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visualized in PBMC but not in HEK cells, supporting the flow cytometry results. Our

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cumulative data revealed that the percentage of CB1-expressing cells within monocytes

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is significantly higher than within lymphocytes (Figure 2D). Mean fluorescent intensity

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(MFI) analysis for probe 3 indicated that the density of CB1 was also significantly

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higher in monocytes than lymphocytes (Figure 2D). Supporting these data, the CB1


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mRNA levels were also significantly higher in purified monocytes than lymphocytes as

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determined by quantitative PCR (Figure 2E). To further confirm the CB1 specificity, we

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stained PBMC with probe 3 and retrieved the obtained positive and negative fractions

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after cell sorting by flow cytometry to quantify the mRNA expression of CB1 (Figure

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2F). Our data demonstrated that the cells contained in the fraction that stain positive for

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probe 3 display significantly higher levels of CB1-specific mRNA than those contained

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in the negative fraction, supporting the correlation between probe 3 fluorescence signal

201

and CB1 expression (Figure 2F). Collectively, all these data demonstrated that HU210-

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Alexa488 probe 3 represents the first fluorescent chemical probe suitable to identify and

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quantify peripheral blood CB1-expressing immune cells by flow cytometry.

204 Lymphocytes

A PBMC

Control

Control

Probe 3

4.1%

59.4%

79.1%

Alexa488

FS

Lymphoid gate

FS

SS

B

C

HEK cells Control

PBMC Control

Probe 3 1.5%

HEK cells Probe 3

Control

Probe 3

2.4%

FS

60

40

20

0

**

800

** 600

400

200 60 40 20 0

** **

150

F *

PBMC

** **

Probe +

100

50

Probe -

SS

0

205 206


1000

CB1 mRNA expression A.u. rel EF1α (x104)

80

** **

ΔMFI after control subtraction

E 100

Alexa488

D

CB1 mRNA expression A.u. rel EF1α (x104)

Alexa488

Monocytes

Probe 3

1.0%

Monocyte gate

% of positive cells after control subtraction

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


*

100

10

1

0 .1

0 .0 1

Probe-

Probe+



Figure 2. Validation of HU210-Alexa488 probe 3 to detect and quantify CB1-expressing

208

immune cells by flow cytometry. Representative flow cytometry dot plots after staining with

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HU210-Alexa488 probe 3 (1 μM) or Alexa Fluor 488 alkyne [2-(6-amino-3-iminio-4,5-

210

disulfonato-3H-xanthen-9-yl)-5-(prop-2-yn-1-ylcarbamoyl)benzoate] (1 μM) as control of (A)

211

gated lymphocytes and monocytes from whole PBMC or (B) HEK cells. (C) Representative

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confocal microscopy images of PBMC and HEK cells after staining with probe 3 or control. (D)

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Percentages and mean fluorescent intensity (MFI) after control subtraction of lymphocytes and

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monocytes in freshly isolated PBMC (n=6 independent experiments) and HEK cells (n=4

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independent experiments) stained positive with probe 3. (E) mRNA expression levels of CB1 in

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sorted lymphocytes, monocytes and HEK cells (n=4 independent experiments) as determined by

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quantitative real-time RT-PCR. Arbitrary units (A.u.) are 2-(ΔCt) values multiplied by 104, with

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ΔCt defined as the difference between the cycle threshold value for CB1 gene and elongation

219

factor 1α (EF1α) as a housekeeping gene. (F) Representative dot plot of the cells stained

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negative or positive with probe 3 in whole freshly isolated PBMC and employed gates for

221

subsequent flow cytometry cell sorting. mRNA expression levels of CB1 in sorted cells stained

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negative and positive with probe 3 (n=6 independent experiments). *P < 0.05 and **P < 0.01.

223

Data represent means with SEMs.

224 225

HU210-Alexa488 probe 3 as a flow cytometry tool to monitor changes in CB1

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expression in blood and tonsil immune cells

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Plasma membrane CB1 decrease following long agonist exposure as a consequence of

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finely regulated mechanisms that shuttle the receptor to lysosomes for degradation,

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contributing to the control of downstream signaling during sustained stimulation.34-36 To

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determine whether the probe 3 could be used to monitor changes in plasma membrane

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CB1, we cultured PBMC in medium or stimulated with the cannabinoid agonists

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WIN552122 or HU210 for 18 h and we tracked CB1 expression with probe 3 (1 μM) by

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flow cytometry (Figure 3A). The percentage of blood lymphocytes expressing CB1 as 12 ACS Paragon Plus Environment

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well as the expression levels of the receptor were significantly downregulated after

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prolonged stimulation with both synthetic cannabinoids (Figure 3A), demonstrating that

236

probe 3 is able to detect up- and down-regulation of plasma membrane CB1 in blood

237

immune cells.

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Next, we isolated tonsil mononuclear cells (TMC) from the tissue following our

239

previously described protocols37 and assessed the capacity of probe 3 to identify tonsil

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CB1-expressing immune cells by flow cytometry (Figure 3B). Tonsils are easily

241

accessible secondary lymph organs located at the main gateway of the respiratory and

242

digestive tracts, representing the first contact point of the immune system with all the

243

pathogens and allergens that enter the body via these ways.12,

244

showed that human tonsils are organs where the induction of oral tolerance occurs

245

through the generation of functional allergen-specific regulatory T (Treg) cells.12, 13, 37

246

Tonsils are organs where immune regulation to viral and bacterial infections,

247

immunometabolism and allergy takes place.39-41 Interestingly, the expression of CB1 at

248

the mRNA level is upregulated in TMC from allergic patients compared to healthy

249

donors.16 The availability of chemical fluorescent probes to track up- and down-

250

regulation of tonsil CB1-expressing cells is of utmost importance to monitor changes in

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the expression of this receptor at the protein level. Our data revealed that probe 3 (1

252

μM) also allows the detection of CB1-expressing cells in TMC (Figure 3B). The

253

percentage of CB1-expressing cells was also significantly downregulated in TMC

254

stimulated with the synthetic cannabinoids WIN552122 or HU210 for 18 h compared to

255

the unstimulated condition (Figure 3B). Collectively, all these data demonstrated that

256

HU210-Alexa488 probe 3 is a suitable tool to monitor physiological changes in the

257

expression pattern of plasma membrane CB1 in blood and tonsil immune cells by flow

258

cytometry.


37, 38

We previously



PBMC

60

** ***

40 20

60

0

*

WIN552122

US

*

60.5%

34.9%

HU210 40.3%

40

Alexa488

80



A

20

0

FS

TMC

B

40

20

0

*

*

50 40

WIN552122

US

* **

43.5%

24.6%

HU210 33.4%

30

Alexa488

60



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20 10 0

FS

260 261 262

Figure 3. HU210-Alexa488 probe 3 allows the tracking of changes in CB1 expression in blood

263

and tonsil immune cells by flow cytometry. Percentages and mean fluorescent intensity (MFI)

264

of (A) PBMC and (B) TMC cultured in medium (US) or with cannabinoid agonists WIN552122

265

or HU210 for 18 h (n=5 independent experiments) stained with probe 3 (1 μM) after control

266

subtraction. Representative flow cytometry dot plots are also displayed in each case. *P < 0.05,

267

**P < 0.01 and ***P

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