**3. Results**

#### *3.1. Cannabinoid Treatments Reduce Nitric Oxide and Cytokine Production of LPS-Activated Peritoneal Macrophages*

Upon activation, macrophages produce large amounts of nitric oxide (NO•). To test the effect of cannabinoid treatments on macrophage activation, NO• secretion was determined. Macrophages from previously thioglycollate (tg)-injected C57BL/6 mice were collected by peritoneal lavage, and then activated for 24 h with lipopolysaccharide (LPS), in the presence of cannabinoid treatments; 1400W dihydrochloride, a specific iNOS inhibitor, served as control. All treatments show dose dependent effect on NO• secretion (Figure S1). For our further experiments, we used 5 μg/mL of each treatment (THC, CBD, or cannabis extracts). Our results demonstrate 42–72% inhibition of activation-induced NO• secretion from peritoneal macrophages from female (Figure 1a) and male (Figure 1b) mice in the presence of cannabinoid treatments. The reduced NO• secretion was not caused by decreasing cell number, since the treatments showed no toxic effect on the cells in MTT viability assay (Figure S2). The differences between THC and CBD treatments were significant only in the female mice. Importantly, both extracts were significantly more efficient (*p* < 0.0001) than the pure cannabinoids in female and male mice. A treatment with the combination of THC and CBD (2.5 μg/mL of each) was less effective than the pure cannabinoids (Figure S3).

Next, we examined the influence of aging on the responsiveness of macrophages to cannabinoid treatments. For this aim, we obtained peritoneal macrophages from aged (18 months old) mice and compared their NO• secretion with macrophages from young (2 months old) mice. Figure 1c (left) shows elevated secretion of NO• from non-activated cells and reduced secretion upon activation of old peritoneal macrophages. The effect of cannabinoid treatments on NO• secretion was significantly reduced as compared with cells from young mice (Figure 1c, right). In addition, peritoneal macrophages from aged mice demonstrate alleviated expression of the cannabinoid receptors (Figure 1d).

**Figure 1.** The influence of pure CBD/THC and cannabis extracts on nitric oxide production of LPS-activated peritoneal macrophages. Peritoneal macrophages from C57BL/6 female (**a**) and male (**b**) mice were activated for 24 h with LPS, in the presence of cannabinoid treatments (5 μg/mL). 1400W (1400), a specific iNOS inhibitor, served as control. NO• levels in the supernatant were analyzed. (**a**)—*n* = 10 mice, from 3 independent experiments; (**b**)—*n* = 7 mice, from 3 independent experiments. The differences of all treatments as compare with LPS-activated control (indicated on the graphs) are highly significant. The differences of THCE and THC between CBDE and CBD are significant in (**<sup>a</sup>**,**b**); (**c**) NO• levels in the supernatant of activated peritoneal macrophages from young (2 months old) and aged (18 months old) C57BL/6 female and male mice. *n* = 7 mice per group, from 3 independent experiments. The differences between aged and young mice in each treatment, are indicated on the graph; (**d**) the expression levels of CB1 and CB2 in peritoneal macrophages from young and aged C57BL/6 female mice (*n* = 3/group) were assessed by real-time PCR analysis. The results are expressed as mean + SEM. *p*-value \*, <0.05; \*\*, <0.01; \*\*\*, <0.001. NA—non-activated; LPS— lipopolysaccharide-activated macrophages; THC—D9 tetrahydrocannabino; CBD—cannabidiol; THCE—high THC cannabis extract; CBDE—high CBD cannabis extract; n.s.—not significant.

To examine the effect of the treatments on inflammatory cytokine/chemokine secretion from peritoneal macrophages, we collected the supernatant of 24 h LPS-activated cells and analyzed the levels of different cytokines using a LEGENDplex™ MU Macrophage/Microglia Panel cytokine array assay. IL6, TNF-alpha, CXCL2, and G-CSF levels in the culture media increased upon activation (Figure 2a–d). Our results demonstrate 40–74% inhibition of activation-induced IL6 secretion from peritoneal macrophages (Figure 2a), 22–66% inhibition of TNF-alpha secretion (Figure 2b), 4–44% inhibition of CXCL2 secretion (Figure 2c), and 0–58% inhibition of GM-CSF secretion (Figure 2d). CBD and CBDE had more significant inhibitory effect on cytokine/chemokine secretion as compared with THC and THCE. The differences between CBD and THC are significant in TNF-alpha, CXCL2, and G-CSF. IL12p40, CCL22, and IL18 were also elevated following activation, however, the cannabinoid-based treatments did not have clear effects on their levels (Figure S4).

**Figure 2.** The influence of pure CBD/THC and cannabis extracts on cytokine/chemokine production of LPS-activated peritoneal macrophages. Peritoneal macrophages were activated for 24 h with LPS, in the presence of cannabinoid treatments (5 μg/mL), as in Figure 1. *n* = 3 mice. IL6 (**a**), TNF-alpha (**b**), CXCL2 (**c**), and G-CSF (**d**) levels in the culture supernatant were detected. The results are expressed as mean + SEM. *p*-value as compare with LPS-activated control cells \*, <0.05; \*\*, <0.01; \*\*\*, <0.001. NA— non-activated; LPS—lipopolysaccharide-activated macrophages; THC—D9 tetrahydrocannabinol; CBD—cannabidiol; THCE—high THC cannabis extract; CBDE—high CBD cannabis extract.

#### *3.2. Cannabinoid Treatments Affect the Phenotype of Activated Peritoneal Macrophages*

To learn more about CBD and THC molecular effects in macrophages, we tested cell surface expression of several molecules in the activated macrophages. Class II molecules of the major histocompatibility complex (MHCII) is upregulated on some polarized macrophage populations upon activation. We found that both THC and CBD treatments induce small, but significant, further elevation in MHCII expression (Figure 3a). R. A. Ezekowitz and S. Gordon have demonstrated that expression levels of the F4/80 glycoprotein and the Fc

receptor CD16/32 on peritoneal macrophages are dependent on the activator [17]. In our study, in tg-induced macrophages activated with LPS, F4/80 was elevated by THC (15%), but reduced by CBD treatment (32%) (Figure 3b). CD16/32 cell surface expression was elevated on LPS-activated cells, but the cannabinoid treatments had no significant effect on its expression (Figure 3c).

**Figure 3.** The effect of CBD and THC on the phenotype of LPS-activated peritoneal macrophages. Peritoneal macrophages from C57BL/6 female mice were activated for 24 h with LPS, in the presence of cannabinoid treatments (5 μg/mL). Cell surface expression levels of MHCII ((**a**), *n* = 5), F4/80 ((**b**), *n* = 6), and CD16/32 ((**c**), *n* = 7) were determined by flow cytometry. The results are expressed as mean + SEM. *p*-value as compare with LPS-activated control cells \*, <0.05; \*\*, <0.01; \*\*\*, <0.001. NA— non-activated; LPS—lipopolysaccharide-activated macrophages; THC—D9 tetrahydrocannabinol; CBD—cannabidiol; n.s.—not significant.

#### *3.3. Cannabis Extracts Have Improved Effect in Murine Colitis DSS Model Mice as Compared with Pure Cannabinoids*

We chose the murine colitis dextran sodium sulfate (DSS) model to compare the efficacy of the different cannabinoid treatments in macrophage-related inflammation in vivo. DSS is a chemical colitogen with anticoagulant properties. In the acute intestinal inflammation model, DSS causes disruption of the intestinal epithelial monolayer lining, leading to the entry of luminal bacteria and associated antigens into the mucosa and activation of the innate immunity [18].

Acute colitis was induced by adding DSS to the mice drinking water at 2% (*w*/*v*) ad libitum for 7 days, and then replaced with plain water. Cannabinoid treatments, 5 mg/kg, were administered intraperitoneal, every other day, from Day 1 to Day 10 (Figure 4a). All treatments, but particularly the CBD extract (CBDE), significantly inhibited weight loss in the DSS mice (Figure 4b). THC and CBD extracts both had better effect on the disease clinical score as compared with the pure cannabinoids (Figure 4c). This improved effect was also evident in the measurement of colon length (Figure 4d); the average length of the colon in the CBDE-treated mice was 4.8 cm, significantly higher than the average length of the colon in the CBD-treated mice 3.9 cm (*p* < 0.0001). In THCE-treated mice, the average length of the colon was 4.8 cm vs. 4.1 cm in the THC-treated mice (*p* < 0.0001). All treatment significantly improved the clinical condition of the mice as compared with the vehicle DSS group. The difference between the CBD and THC groups was not significant.

**Figure 4.** Cannabis/cannabinoids administration for treatment of murine colitis in DSS model mice: (**a**) Acute colitis was induced by adding DSS to the mice drinking water at 2% (*w*/*v*) ad libitum for 7 days,

and then replaced with plain water. 5 mg/kg cannabis/cannabinoids were administered IP every other day, starting from Day 1; (**b**) the effect of pure cannabinoids (left) and cannabis extracts (right) on weight loss. Differences between DSS group and THCE- as well as CBDE-treated groups are significant. The difference between the CBD- and CBDE-treated groups is significant *p* < 0.05; (**c**) the effect of pure cannabinoids (left) and cannabis extracts (right) on clinical score. Differences between the DSS group and all cannabinoid-treated groups are significant *p* < 0.005. Differences between cannabis extracts and pure cannabinoids are significant *p* < 0.008. The difference between the CBDand THC-treated groups is significant *p* < 0.05; (**d**) the effect of pure cannabinoids and cannabis extracts on colon length, at the end of the experiment (Day 10). *p*-value as compare with the DSS control group \*\*\*, <0.001. The differences between THCE and THC and between CBDE and CBD are significant. Data are summarized from 5 independent experiments, 5–7 mice/group in each experiment. DSS—dextran sodium sulfate; THC—D9 tetrahydrocannabinol; CBD—cannabidiol; THCE—high THC cannabis extract; CBDE—high CBD cannabis extract.

The improved clinical outcome of all cannabinoid-based treatments is also demonstrated in the histopathology of the colon (Figure 5a,b). Colon sections were stained with haematoxylin/eosin and scored for the infiltration with inflammatory cells, damage in cryp<sup>t</sup> architecture, and thickening of the bowel wall. In this assay, no significant differences between the four cannabinoid-based treatments were found.

**Figure 5.** Histopathology of the colons of DSS model mice. Paraffin sections were stained with H&E and scored for inflammation and tissue damage; (**a**) Summary of average scores. *p*-value as compare

with the DSS control group \*\*, <0.01; \*\*\*, <0.001; (**b**) representative pictures of H&E stained sections. DSS—dextran sodium sulfate; THC—D9 tetrahydrocannabinol; CBD—cannabidiol; THCE—high THC cannabis extract; CBDE—high CBD cannabis extract.

#### *3.4. Cannabinoid Treatments Reduce Intestinal Macrophage Infiltration and the Levels of Inflammatory Cytokines in the Plasma of DSS Mice*

Next, we examined the effects of the treatments on macrophages and inflammation in the DSS model. In IBD patients, an increased number of macrophages in the inflamed mucosa initiate a rapid response to luminal microbial antigens [3]. Immunohistochemistry with F4/80 antibody was performed to detect macrophages in the colon. As demonstrated in Figure 6a,b, the number of macrophages in the colon tissue is highly elevated in the DSS mice. All the cannabinoid-based treatments significantly inhibited colon infiltration of macrophages in the DSS mice (40–60% inhibition). CBDE treatment inhibition of macrophage infiltration was significantly more effective than pure CBD treatment (*p* = 0.03). The THCE and THC treatments were not significantly different.

**Figure 6.** Immunostaining for macrophages in the colons of DSS model mice. Paraffin sections were stained with anti-F4/80 antibodies: (**a**) Average number of positive cells. *p*-value as compare with the

DSS control group \*, <0.05; \*\*, <0.01; \*\*\*, <0.001. The difference between the CBDE and CBD groups is significant; (**b**) representative pictures of F4/80 immunostained sections. DSS—dextran sodium sulfate; THC—D9 tetrahydrocannabinol; CBD—cannabidiol; THCE—high THC cannabis extract; CBDE—high CBD cannabis extract.

Cytokine levels in the blood reflect the inflammatory status in the body. It was also demonstrated that inflammatory cytokines have a crucial role in the pathogenesis of IBD, where they control multiple aspects of the inflammatory response [19]. We, therefore, tested the plasma levels of inflammatory cytokines of treated DSS mice using a mouse inflammation cytokine array assay. Interestingly, we found that the different treatments have unique effects on plasma cytokines (Figure 7). IL6 levels were reduced by all treatments, but particularly by CBDE. The levels of TNF-alpha were significantly reduced only by the cannabis extracts; but pure cannabinoids had stronger effect on IFN-beta levels. The levels of other tested cytokines were not significantly changed in the plasma of DSS mice as compared with healthy controls.

**Figure 7.** The influence of pure CBD/THC and cannabis extracts on inflammatory cytokines in the blood of DSS model mice. Blood samples for cytokine analysis were obtained at Day 10. The levels of TNFa (**a**), IL6 (**b**), and IFNb (**c**) in the plasma were determined using a LEGENDplex ™ Mouse Inflammation Panel cytokine array; *n* = 5–10 mice/group. The results are expressed as mean + SEM. *p*-value as compare with the DSS group \*, <0.05; \*\*, <0.01; \*\*\*, <0.001. DSS—dextran sodium sulfate; THC—D9 tetrahydrocannabinol; CBD—cannabidiol; THCE—high THC cannabis extract; CBDE—high CBD cannabis extract; n.s.—not significant.
