**4. Discussion**

Macrophages play key roles in innate immunity. They are specialized cells involved in the detection and destruction of bacteria and other harmful pathogens. In addition, they can present antigens to T cells and initiate inflammation. Macrophages and microglial cells express the Gi protein-coupled seven transmembrane cannabinoid receptors, CB1 and CB2 [12,20–24]. On the one hand, CB2 receptors in macrophages have anti-inflammatory properties, can affect macrophage polarization, and are involved in promoting autophagy [3,5,6,8]. CB1 receptors in macrophages, on the other hand, have proinflammatory properties and are involved in phagocytosis [9–12].

Phytocannabinoids possess a wide range of immune regulatory properties, mediated by the endocannabinoid system [4]. Two cannabinoids have been the focus of most of the studies examining medical uses: THC and CBD. These two phyto-cannabinoids utilize different endocannabinoid receptors to mediate their effects. A few studies have demonstrated anti-inflammatory effect for phytocannabinoid treatments on macrophage function, however, these studies have generally focused on a single cannabinoid [25–29].

Previously, we compared the influence of THC, CBD, and cannabis extracts on lymphocyte activation in vitro and in a murine graft versus host disease (GvHD) model [14]. In the current research, we aimed to compare the consequences of treatment with THC or CBD on macrophage activation and in DSS murine model for gastrointestinal inflammation. We use this model as a model for acute inflammation with involvement of macrophages in the pathophysiology of the disease [19].

First, we examined the effect of cannabinoid treatments on NO• secretion from peritoneal macrophages, ex vivo. Macrophages produce large amounts of NO• as a defense mechanism. However, in pathological conditions, such as autoimmune diseases, there is excessive simultaneous production of NO• and O2•− by macrophages and other cells. NO• and O2•− generate large quantities of the toxic molecule peroxynitrite (ONOO −), an oxidant and nitrating agen<sup>t</sup> which can damage a wide array of molecules in cells, including DNA and proteins [30]. Elevated NO• bioavailability has been implicated in the etiology of a number of pathological events including IBD. It is now clear that the ECS plays a key role in regulating NO• formation via CB1R, CB2R, and/or alternative molecular targets [31]. We found that both THC and CBD, as well as cannabis extracts treatments, inhibit activation-induced NO• secretion from both female and male peritoneal macrophages. These results were in agreemen<sup>t</sup> with the results of Romano et al. who demonstrated a similar effect with the cannabinoid THCV [32]. Cannabis extracts and pure cannabinoids were used in a concentration of 5 μg/mL; the extracts had stronger effect than the pure cannabinoids, although cannabinoids constituted only 35–38% of their content. This could result either from inhibitory signaling of other molecules in the plant (not THC/CBD) or from a synergistic function of THC/CBD with other molecules. Interestingly, CBD signaling has been associated with inhibition of cytokine/chemokine secretion, since both pure CBD and CBDE had greater effect on the levels of peritoneal macrophage's secreted cytokines as compared with THC and THCE. It should be noted that the cannabis extracts had no significant advantage over the pure cannabinoids in inhibition of cytokine/chemokine secretion from peritoneal macrophages. The inhibitory effect of CBD, on the one hand, IL6 and TNF-alpha, and on the other hand, CXCL2 and G-CSF may sugges<sup>t</sup> its ability to regulate both local inflammation and periphery bone marrow interactions.

Aging is concurrent with a slow and constant functional deterioration of the immune system, known as immunosenescence, which is accompanied by an increase in chronic inflammatory processes, a phenomenon known as "inflammaging". The devastating consequences of an aged immune system include impairment of the ability to cope with infections and an increased risk of developing chronic diseases such as cancer and heart disease—the two leading causes of death in old age [33]. Indeed, our results demonstrate elevated NO• secretion from non-activated peritoneal macrophages from aged mice, which indicated pre-existing inflammatory process. In addition, the reduced secretion upon LPS activation of aged mice peritoneal macrophages, may indicate immunosenescence. The old cells also showed decreased responsiveness to cannabinoid treatments and alleviated expression of CB1 and CB2 receptors. These results corresponds with earlier studies which demonstrated age-related alterations in the endocannabinoid system [34,35], and may be accompanied with other age-related changes in the endocannabinoid receptors and enzymes in the cells.

The effect of THC and CBD treatments on the expression of cell surface molecules on activated peritoneal macrophages was examined. Stimulation with LPS, a major component of the outer membrane of Gram-negative bacteria, leads to an effective classical, proinflammatory, macrophage activation. MHC-II molecules, expressed by antigen presenting cells,

are central in the initiation of cellular and humoral immune responses. Upon LPS activation, MHCII is upregulated in macrophages [36], and hence increased antigen presentation. MHCII levels can also be affected by different soluble factors, such as cytokines [37]. In a previous study by Paul W. Wacnik et al. THC reduced MHCII expression in LPS-activated dendritic cells [38]. Since cannabinoid treatments reduced nitric oxid secretion, we expected similar reduced expression of MHCII. Surprisingly, we found that both treatments did not reduce, but induced, a small elevation of MHCII expression in the activated macrophages. The combination of these results may indicate that treatment of cannabinoids together with LPS may lead to M2b polarization of the peritoneal macrophages. M2b macrophages are a population of regulatory macrophages, classically induced by LPS and immune complexes, G-protein coupled receptor ligands, or prostaglandins [39,40].

F4/80 is widely used as a mouse macrophage antigen marker. Although the ligands for F4/80 have not been defined, evidence shows that this molecule is required for peripheral tolerance [41]. In addition, it has been demonstrated that F4/80 and Fc receptor CD16/32 expression levels in peritoneal macrophages are dependent on the activator [17]. In our experiments, the expression of F4/80 was elevated by THC, but reduced by CBD treatment as compared with the activated control cells. This result demonstrates the different consequences of the treatment with different cannabinoids. However, the functional relevance of this result is ye<sup>t</sup> to be determined. CD16 and CD32 are low affinity IgG Fc receptors. Immune complexes signaling through the Fc receptors can lead to M2B polarization [39]. In our experiments, CD16/32 cell surface expression was elevated in LPS-activated cells, however, the cannabinoid treatments had no significant effect on their expression. Therefore, it is possible that the cannabinoids bypass CD16/32 signaling, for example, by binding to PPARγ, which may provide the second signal for M2B polarization.

IBD is comprised of well-known gastrointestinal autoimmune inflammatory disorders such as ULcerative colitis (UC) and Crohn's (CD). Cannabinoid receptor stimulation attenuates murine colitis, while cannabinoid receptor antagonism and cannabinoid receptor deficient models reverse these anti-inflammatory effects [4]. Phytocannabinoids have been used in preclinical models of gastrointestinal inflammation [42,43] and several clinical trial have tested the efficacy of cannabinoid-based treatments in IBD patients [42,44]. Indeed, in our DSS murine colitis model, cannabinoid treatments improved the clinical condition of the mice. Similar to the in vitro nitric oxide secretion assay and to our previous results in the murine GvHD model [14], the results of treatment with cannabis extracts were superior to pure-cannabinoid treatments. This unique effect could be a result of either a synergistic function of THC/CBD with other components or from independent anti-inflammatory properties of other molecules in the plant.

Dysregulated macrophages have a key role in the pathogenesis of IBD. In IBD patients, the number of macrophages increases in the inflamed mucosa. The phenotype and functions of the macrophages in the inflamed sites differ from those in physical conditions. For instance, they express high levels of costimulatory molecules and produce high levels of IL-12 and IL-23 in vitro under the microbial stimulation [3]. Our results demonstrate that cannabinoid-based treatments inhibit colon infiltration of macrophages in DSS mice. The average number of colon infiltrating macrophages in each treatment group correlated with the severity of the disease.

Inflammatory cytokines are produced by macrophages, but also by other cells such as dendritic cells, fibroblasts, and lymphocytes. The cannabinoid treatments all affected the levels of inflammatory cytokines in the mice blood. However, each treatment had a unique effect on the composition of cytokines: CBD treatment reduced the levels of IL6 and the more regulatory cytokine, interferon-beta; THC treatment reduced the levels of IL6 and interferon-beta better than CBD; CBDE treatment reduced the levels of TNF-alpha and had the strongest effect on the level of IL6; and THCE treatment reduced the levels of IL6, interferon-beta; and TNF-alpha. These results demonstrate, once again, the different consequences of treatment with different cannabinoid-based treatments. Interestingly, IL6, a key cytokine in IBD [19] was reduced by all the cannabinoid treatments, but TNF-alpha,

another cytokine involved in the pathogenesis of IBD, was significantly affected, in the murine model, only by the cannabis extracts. Therefore it is possible that the reduced levels of TNF-alpha are responsible for the improved clinical effect. Importantly, anti-TNF agents are often used for IBD treatment in the clinic, due to the central role of this cytokine in the pathogenesis of the disease in human subjects too. CBDE was the most effective treatment in IL6 and TNF-alpha inhibition, both in activated peritoneal macrophages and in the murine model. This result was in agreemen<sup>t</sup> with the recently published results of Aswad et al., who demonstrated similar effects for a different high-CBD cannabis extract in human PBMCs and neutrophils and in murine models for systemic and lung inflammation [45].
