**1. Introduction**

Macrophages are specialized innate immune cells that orchestrate homeostatic, inflammatory, and reparative activities. Murine macrophages are located in the brain, skin, liver, kidney, lungs, and heart and originate from the yolk sac or fetal liver; their maintenance in adulthood in the absence of stressors is independent of circulating monocytic precursors. In other tissues, such as the gastrointestinal tract, monocytic precursors contribute to tissue macrophages [1]. In the steady state, tissue macrophages have intrinsic anti-inflammatory functions. Tissue stress, including infection, drives the production of monocytes and neutrophils. Bone marrow-derived monocytes are recruited to the damaged site, differentiate into macrophages and dendritic cells, and begin the inflammatory processes [2]. These events must be tightly regulated. Dysregulation of macrophage differentiation and function is involved in the pathogenesis of different diseases, including inflammatory bowel disease (IBD).

Immunological dysregulation in IBD is characterized by epithelial damage, expansion of inflammation driven by intestinal flora, a large number of cells infiltrating into the lamina propria, and a failure of immune regulation to control the inflammatory response [3]. In IBD patients, the number of macrophages increase in the inflamed mucosa [3].

The endocannabinoid system (ECS) regulates various aspects of physiological, behavioral, immunological, and metabolic functions. It is now clear that many of the components of the endocannabinoid system function as key regulators of the immune system and the immune response [4]. Endocannabinoid ligands and receptors are involved in the regulation of both innate and adaptive immune cells. Murine and human monocytes/macrophages and microglial cells express the endocannabinoid receptors CB1 and CB2. CB2 receptors in macrophages have anti-inflammatory properties [5–8], while CB1 receptors have proinflammatory properties and are involved in phagocytosis [9–12]. Importantly, the expression levels of cannabinoid receptors in leukocytes are influenced by different inflammatory factors [13].

Phytocannabinoids, the biologically active constituents of cannabis, possess a wide range of immune regulatory properties, mediated by the endocannabinoid system. Two cannabinoids have been the focus of most of the studies that have examined medical uses, i.e., D9 tetrahydrocannabinol (THC) and cannabidiol (CBD). THC and some of the other phytocannabinoids mediate their biological effects primarily through the classical cannabinoid receptors CB1 and CB2. In addition, THC can act as an agonist of the receptors/channels GPR55, GPR18, PPARγ, transient TRPA1, TRPV2, TRPV3, and TRPV4, and as an antagonist of the receptors/channels TRPM8 and 5-HT3A. Interestingly, although CBD affects the immune function, it has a very weak affinity to CB2 or CB1, where it can act as a negative allosteric modulator. Several reports have demonstrated that CBD acts as an agonist of other receptors/channels, such as TRPA1, TRPV1, TRPV2, TRPV3, PPARγ, and 5-HT1A, and as an antagonist of the receptors GPR55, GPR18, and 5-HT3A. CBD is also an inverse agonist of the receptors GPR3, GPR6, and GPR12 [4].

Previously, we compared the influence of cannabinoid-based treatments on lymphocyte function [14]. The aim of the current research was to examine the consequences of treatment with THC and CBD on macrophage activation and in macrophage-related inflammation. Since THC and CBD mediate their actions on mammal cells though different receptors, we hypothesized that each cannabinoid has selective effects on macrophage phenotype and function, and hence, a different impact on activation and inflammation. Therefore, the aim of our research was to elucidate the differential effects of THC- and CBD-based treatments on macrophage immune function. Our previous results suggested that the combination of cannabinoids with other active molecules in the plant may achieve better clinical results than pure cannabinoids, therefore, we also examined the differences between the effects of high THC and high CBD cannabis extracts (Table 1).


**Table 1.** Chemical analysis of the main phytocannabinoids and terpenoids in the cannabis extracts.

While Cannabis is not ye<sup>t</sup> registered as a drug, the potential of cannabinoid-based medicines for the treatment of various conditions has led many countries to authorize their clinical use. As a result, in recent years, there has been a rapid increase in the medical use of cannabis and a wide range of cannabinoid-based treatments are offered to patients. THC and CBD are considered to be the two essential elements in these treatments. Therefore, it is

crucial to explore the various biological effects of these molecules. A better understanding of the effects of THC, CBD, and other active molecules on the immune response will assist physicians in providing the best possible individually targeted treatment for their patients and will allow the design of new treatments.

#### **2. Materials and Methods**

#### *2.1. Cannabis Extracts and Cannabinoids*

This research was performed under the approval of The Medical Cannabis Unit in the Israeli Ministry of Health (REQ46). Pure THC was generously provided by the laboratory of Prof. Raphael Mechoulam. Synthetic CBD was purchased from STI Pharmaceuticals Ltd., Newtown, UK. Cannabis Sativa and Indica extract with high content in THC or CBD (i.e., THCE/CBDE, respectively) were supplied by Cannabliss (Cannabliss Ltd., Tel Aviv, Israel). Extraction was obtained using ethanol, and evaporated. Identification and quantification of phytocannabinoids in the cannabis extracts were done by ultrahigh performance liquid chromatography with an ultraviolet detector (UHPLC/UV) system (Thermo Scientific, Bremen, Germany). The terpenoid analysis was performed by static headspace gas chromatography tandem mass spectrometry (SHS-GC/MS/MS) using full evaporation technique with external calibrations, as previously described [15,16]. The main molecules are listed in Table 1.
