High-CBD Extract (CBD-X) in Asthma Management: Reducing Th2-Driven Cytokine Secretion and Neutrophil/Eosinophil Activity

2.1. CBD-X Extract Impairs Differentiation of Human CD4 T Cells into Th2 Cells and Downregulates IL-5 and IL-13 Secretion

Based on our previous research on the effects of CBD-X extracts from the High CBD- cannabis strain, on cytokine storms and lung disease [30], we were prompted to investigate its impact on chronic lung diseases such as asthma. Th2 cells play a critical role in asthma development [31], secreting the pro-inflammatory type 2 cytokines IL-5 and IL-13 [6,7]. Moreover, Th2 cells differentiate from CD4 T cells in response to cytokines such as IL-4 [32].

We first analyzed the effect of CBD-X extract on the differentiation of CD4 T cells into Th2 cells ex vivo. Isolated CD4 T cells were treated with the high-CBD extract CBD-X. The cells were then differentiated into Th2 cells over six days. Cells were subjected to flow cytometry analysis using CD4, CCR4, and CCR6 markers. The percentage of CCR4+CCR6- in the CD4 population, termed Th2 cells, was determined.

Differentiated Th2 cells showed a fourfold increase in the percentage of CCR4+CCR6- cells compared to inactivated cells. However, CBD-X significantly reduced the capacity of CD4 T cells to differentiate into Th2 cells back to baseline levels (Figure 1a). The flow cytometry analysis demonstrated a marked decrease in the proportion of Th2 cells among CBD-X-treated CD4 T cells compared to the differentiated control group. Representative results of Th2 cell flow cytometry analysis are shown in three groups: inactivated, differentiated, and CBD-X differentiated cells (Figure 1b).

Given that Th2 cells secrete IL-5 and IL-13 during asthma exacerbation, we further examined the effect of CBD-X extract on the secretion of these cytokines. Differentiated Th2 cells significantly increased the secretion of IL-5 and IL-13 by threefold, compared to the control. However, CBD-X-treated Th2 cells exhibited a substantial reduction in the levels of these cytokines (Figure 1c,d).

To exclude the possibility that this observation was due to a cytotoxic effect of the added extract, cell viability was measured via Alamar Blue assay. Cell viability was not reduced with the addition of the cannabinoid samples, allowing us to conclude that the reduction in differentiation and cytokine secretion was due to a regulatory effect of CBD-X (Figure S1).

These results indicate that CBD-X extract not only impairs the differentiation process but also diminishes the functional capacity of Th2 cells, potentially impacting Th2-mediated immune responses in chronic lung diseases like asthma.

2.2. CBD-X Extract Attenuates the Secretion of Pro-Inflammatory Cytokines from PBMC Derived Neutrophils

Some asthma patients exhibit elevated neutrophil counts in their airway secretions [33], which often show resistance to steroid treatments [34]. Neutrophilia in asthma is linked to factors such as IL-8, IL-17, and granulocyte-colony stimulating factor (G-CSF). The production of IL-8 is increased in some asthma patients [35,36], and clinical data reveal that IL-8 can serve as a marker for type 2-low asthma [37]. IL-8 is a crucial chemokine responsible for recruiting and activating neutrophils. Additionally, it is secreted by activated neutrophils, further amplifying the inflammatory response. To further investigate the role of CBD-X extract in this context, we examined its effect on neutrophils. Isolated human-derived neutrophils were pre-treated with 2 mg/mL CBD-X extract or vehicle as a control and then activated with 100 ng/mL lipopolysaccharide (LPS) overnight to induce cytokine secretion. The levels of IL-8 and IL-6 were analyzed. Our results demonstrate that LPS significantly increased the release of the pro-inflammatory cytokines IL-8 and IL-6 by about fourfold compared to the control (Figure 2a,b). However, CBD-X-treated neutrophils exhibited a significantly reduced capacity to secrete both cytokines. It is worth noting that the viability of neutrophils was not affected by the CBD-X extract at the concentration used, as determined via the Alamar Blue assay (Figure S2).

2.3. CBD-X Extract Inhibits IL-8 Induced Migration of Neutrophils

Neutrophils are recruited to the airways of asthma patients in response to various chemokines, with IL-8 being particularly crucial for their recruitment [38,39]. IL-8 plays a significant role in asthma pathology by promoting increased neutrophilic infiltration into the lungs and exacerbating lung damage [40]. To determine whether CBD-X extract affects the migration capacity of neutrophils towards IL-8, PBMC-derived neutrophils were treated with 1 or 2 µg/mL CBD-X extract. Their ability to migrate towards IL-8 across a Boyden chamber was tested. Cells that migrated through the pores into the lower chamber were collected and subjected to flow cytometry analysis. IL-8 induced neutrophil migration significantly compared to the control. However, CBD-X extract inhibited the migration of neutrophils towards IL-8 in a dose-dependent manner (Figure 3). Neutrophil migration was inhibited by 2.5-fold and 3.5-fold with 1 and 2 µg/mL CBD-X extract, respectively (Figure 3a,b), effectively attenuating the IL-8 induced migration capacity.

These results, combined with the observed downregulation of IL-8 and IL-6 secretion by CBD-X-treated neutrophils, suggest that CBD-X may modulate the inflammatory response by impairing both neutrophil recruitment and activity. This could potentially offer a therapeutic strategy for managing conditions characterized by excessive neutrophilic inflammation, such as type 2-low asthma.

2.4. CBD-X Extract Modulates IgE Levels and Cytokine Secretion in a Murine Model of Asthma

We investigated the therapeutic potential of CBD-X extract in an in vivo mouse model of asthma. BALB/c mice were sensitized with ovalbumin (OVA, grade V) to induce an allergic response. To assess the effects of CBD-X extract, lung fluids and blood were collected and analyzed (Figure 4).

We first quantified serum IgE levels, given its role as a marker of allergic asthma [41]. The secretion of OVA-IgE levels in the serum was detected via ELISA. The increased levels of OVA-IgE in the serum of OVA-treated mice were reduced by more than twofold with the CBD-X extract (Figure 4a).

Next, we analyzed cytokine levels in the bronchoalveolar lavage fluid (BALF) from the lungs. Remarkably, the CBD-X extract led to a substantial reduction in the pro-asthmatic cytokines IL-4, IL-5, and IL-13 compared to the OVA-treated mice (Figure 4b–d).

These results indicate that CBD-X extract effectively attenuates both systemic and localized inflammatory responses associated with allergic asthma.

2.5. CBD-X Extract Inhibits Migration of Leukocytes to OVA-Induced Asthmatic Lungs

The asthmatic airway is characterized by chronic inflammation of the airway wall due to a complex infiltration and interplay of immune cells [11]. Therefore, we examined whether CBD-X extract affected leukocyte migration into the lungs in an OVA-treated mouse asthma model. Lung fluids were collected and immune cells were marked with anti-mouse APC-CD45, anti-mouse FITC-F4/80, anti-mouse BV786-LY6G, and PE-Siglec F for flow cytometry analysis.

Examination of lung immune cell populations revealed that control mice with asthma (OVA-treated mice) exhibited elevated numbers of leukocytes, and eosinophils, while neutrophil and macrophage numbers remained unchanged (Figure 5). In contrast, CBD-X-treated mice showed a significant reduction in leukocyte and eosinophil counts by 51% and 58%, respectively (Figure 5a), suggesting a targeted attenuation of the inflammatory response. Macrophage and neutrophil numbers were unaffected, indicating that CBD-X extract modulates specific immune cell populations rather than broadly suppressing immune activity.

The gating strategy of representative results is shown (Figure 5b–e). Collectively, these findings demonstrate that CBD-X treatment effectively alleviates asthma symptoms in the mouse model. The reduction in IgE levels and pro-inflammatory cytokines, along with the decreased recruitment of inflammatory cells, underscores the potential of CBD-X extract as a therapeutic agent for asthma, targeting both systemic and localized inflammatory processes.

4.1. Reagents

ELISA kits for human IL-5, IL-13, IL-8, and IL-6 or mouse IL-4, IL-5, and IL-13 were obtained from R&D System (Minneapolis, MN, USA). The Mouse Anti-OVA IgE Antibody Assay Kit was obtained from Chondrex, Inc (Woodinville, WA, USA). Human TruStain FcX™ (Fc Receptor Blocking Solution), anti-human CD3 (mouse, IgG2a, κ, clone OKT3), anti-human CD28 (mouse, IgG1, κ, clone CD28.2), human IL-2 and anti-human IFN-γ (Mouse IgG1, κ, clone B27), APC anti-human CD4, (mouse, IgG2b, κ, clone A17070D), Brilliant Violet 421 anti-human CD194 (CCR4, Mouse IgG1, κ, clone L291H4) and FITC anti-human CD196 (CCR6, Mouse IgG2b, κ, clone G034E3), TruStain FcX™ (rat anti-mouse CD16/32, IgG2a, Λ, a) and APC anti-mouse CD45 (rat, IgG2b, κ, clone 30/F11), FITC anti-mouse F4/80 recombinant (Mouse IgG1, κ, clone QA17A29), Brilliant Violet 785 anti-mouse Ly-6G (Rat IgG2a, κ, clone 1A8), and PE anti-human Siglec-8 (Mouse IgG1, κ, clone 7C9) were purchased from BioLegend (San Diego, CA, USA). The media RPMI 1640 Medium with L-Glutamine were obtained from Sartorius, Beit Haemek, Israel and the medium X-VIVO 15 with gentamicin and phenol red was obtained from Lonza (Basel, Switzerland). The Human Neutrophil Isolation Kit and the Human CD4+ T -Cell Isolation Kit were obtained from STEMCELL Technologies, Vancouver, BC, Canada. Millicell Hanging Cell Culture Inserts, PET 3 µm, and the 24-well were purchased from Merck (Rahway, NJ, USA). Lipopolysaccharide (LPS) was obtained from Santa Cruz (CA, USA). Human IL4 and IL8 were obtained from PeproTech (London, UK). Fetal bovine serum (FBS), glutamine and penicillin, and 100 U/mL streptomycin were purchased from Biological Industries, Beit Haemek (Israel). Albumin from chicken egg white and lyophilized powder (Ovalbumin) were purchased from Merck (Rahway, NJ, USA). Imject™ Alum Adjuvant was purchased from Thermo Fisher Scientific (Waltham, MA, USA).

4.2. Cannabis Extracts

Cannabis extracts were kindly provided by Raphael Pharmaceutical, Inc (Las Vegas, NV, USA). The strains were cultivated and grown by WOLC-Way Of Life Cannabis (Israel). A concentration of 1 µg/mL CBD-X contained 35% CBD, 0.3% THC, 0% CBN, and 0.3% CBG.

4.3. Human Peripheral Blood Samples

Human peripheral blood samples were provided by the Israeli National Biobank for Research (MIDGAM) at Rambam Health Care Campus. The experiments were authorized by the Helsinki Committee at Rambam Health Care Campus (Authorization No. 0442-20 RMB).

4.4. Mice

BALB/c mice at the age of 8 to 10 weeks were purchased from Envigo, Israel. All mice were housed at a barrier-free and specific pathogen-free animal facility at the Pre-Clinical Research Authority, Technion-Israel Institute of Technology in Haifa, Israel. All experiments were performed according to the regulations of the Inspection Committee on the Constitution of the Animal Experimentation of the Technion-Israel Institute of Technology in Haifa, Israel from which authorization for performing animal studies was approved (Authorization No. IL-1330221). Experiments conformed to the regulations in the Prevention of Cruelty to Animals Law (Experiments on Animals) 5754-1994 and the Prevention of Cruelty to Animals Rules (Experiments on Animals) 5761-2001 (correct as of 1 December 2005).

4.5. Isolation of Immune Cells from Blood

Peripheral blood samples of healthy volunteers were collected in EDTA-containing tubes.

4.6. Cell Culture and Treatment of the Cells with Cannabis Extracts

4.7. Cell Viability Measurement

CBD-X-treated Th2 or neutrophils were washed, and medium was added to the cells with 10% Alamar Blue solution. As a negative control, Alamar Blue was added to the medium without cells. The cells were further incubated for another four hours at 37 °C. The absorbance of the test and control wells was read at 570 nm and 600 nm with a standard spectrophotometer [30].

4.8. Cell Migration

For the cell migration experiments, 3 µm 24-well Boyden chambers were used. A total of 0.25–0.5 × 10⁶ cells/mL of neutrophils were seeded in the upper chamber of the 24-well Boyden chamber in medium. In the lower chamber, 100 ng/mL of hIL-8 were placed. Increased concentrations of cannabis extract (1 and 2 μg/mL) or DMSO as a vehicle functioning as a negative control were added to the cells. Three hours later, cells that were migrated through the pores into the lower chamber were collected and counted via flow cytometry [30].

4.9. Asthma Mouse Model

BALB/c mice were intraperitoneally (i.p) injected with 100 µg albumin, chicken egg grade V (OVA), 3 mg/mL Inject Alum (aluminum hydroxide (40 mg/mL), and magnesium hydroxide (40 mg/mL) on days 1, 7, and 14 [47,48]. Then, mice were challenged intranasally (i.n) with 150 µg OVA or PBS on days 21, 23, and 26. Mice were treated intraperitoneally with 300 mg/kg CBD-X extract or vehicle (5% kolliphor, 5% ethanol, 90% of 0.9% NaCl in sterile water for injections) as a control on days 20, 21, 23, and 26 (Figure 6). After 3 h of the last challenge, the mice were euthanized. Then, lung fluids were collected using PBS through lavage and blood was drained from the heart. Samples were centrifuged and supernatants were collected and stored at −20 °C. Levels of OVA-IgE were detected in the serum and cytokines of IL-4, IL-5, and IL-13 levels were detected in the lung fluids via ELISA. Moreover, cells in the lung fluids were stained with TruStain FcX™ (anti-mouse CD16/32) and anti-mouse APC-CD45, anti-mouse FITC- F4/80, anti-mouse BV786-LY6G, and PE- Siglec F for flow cytometry analysis.