High-CBD Extract (CBD-X) Downregulates Cytokine Storm Systemically and Locally in Inflamed Lungs

Abstract

Cytokine storm refers to the dysregulated production of inflammatory mediators leading to hyperinflammation. They are often detrimental, and worsen the severity of COVID-19 and other infectious or inflammatory diseases. Cannabinoids are known to have anti-inflammatory effects but their possible therapeutic value on cytokine storms has not been fully elucidated. In vivo and ex vivo studies were carried out to investigate the effects of high-THC and high-CBD extracts on cytokine production in immune cells. Significant differences between the extracts were observed. Subsequent experiments focusing on a specific high CBD extract (CBD-X) showed significant reductions in pro-inflammatory cytokines in human-derived PBMCs, neutrophils and T cells. In vivo mouse studies, using a systemically inflamed mouse model, showed reductions in pro-inflammatory cytokines TNFα and IL-1β and a concurrent increase in the anti-inflammatory cytokine IL-10 in response to CBD-X extract treatment. Lung inflammation, as in severe COVID-19 disease, is characterized by increased T-cell homing to the lungs. Our investigation revealed that CBD-X extract impaired T-cell migration induced by the chemoattractant SDF1. In addition, the phosphorylation levels of T cell receptor (TCR) signaling proteins Lck and Zap70 were significantly reduced, demonstrating an inhibitory effect on the early events downstream to TCR activation. In a lung inflamed mouse model, we observed a reduction in leukocytes including neutrophil migration to the lungs and decreased levels of IL-1β, MCP-1, IL-6 and TNFα, in response to the administration of the high-CBD extract. The results presented in this work offer that certain high-CBD extract has a high potential in the management of pathological conditions, in which the secretion of cytokines is dysregulated, as it is in severe COVID-19 disease or other infectious or inflammatory diseases.

Keywords: CBD - cannabidiol; COVID - 19; cannabis; corona; cytokine storm and inflammation; lung inflammation.

Figure 1

CBD-X downregulates the secretion of pro-inflammatory cytokines. (A) Mouse macrophages (RAW 264.7 cells) were incubated in DMEM overnight and treated with different cannabinoid extracts at equal concentrations of the respective cannabinoid. Incubation lasted for three hours. High THC-extracts were termed THC-A, THC-B, THC-C and high CBD-containing extracts were termed CBD-X, CBD-Y and CBD-Z. Medium containing cannabinoids was discarded, and cells were incubated with 0.5 µg/ml LPS overnight. IL-6 cytokine levels were detected by ELISA. Means were calculated and normalized to DMSO treated cells. Error bars represent the standard deviations of the means of three biologically independent experiments. (B, C) Neutrophils were isolated from the blood of healthy donors by negative magnetic selection with the EasySep Direct Human Neutrophil Isolation Kit. Isolated neutrophils were treated with 2µg/ml CBD-X or DMSO (vehicle) as a control for two hours. Treated cells were activated by 100 ng/ml LPS overnight. Levels of IL-6 (B) and TNF-α (C) were detected by ELISA. Each colored dot represents one donor. The means were calculated from healthy donors (black line) and each dot represents one case. Data were normalize in ratio to "LPS, DMSO" group and analyzed by one-way ANOVA (Fisher's LSD test with values p < 0.05 considered statistically significant, (***p < 0.001).

Figure 2

CBD-X extract attenuates the secretion of pro-inflammatory cytokines from activated human PBMC-derived T cells or CD4⁺ T cells. PBMCs were isolated from healthy donor blood and were treated with CBD-X or DMSO as a control in RPMI 1640 for two hours. Then, anti-CD28 and anti-IL-2 were added to the cells and cells were transferred to a cell culture plate coated with anti-CD3, for three days. Cells were centrifuged, supernatants were collected, and levels of pro-inflammatory cytokines TNF-α (A, D), IL-6 (B) and IFN-γ (C, E) were detected by ELISA. (A–C) Secreted cytokines from PBMCs are shown. (D, E) Cytokines from CD4⁺ T cells are shown. The means were calculated from healthy donors (black line) and each dot represents one case. Data were normalized in ratio to activated cells treated with DMSO group and analyzed by one-way ANOVA (Fisher's LSD test with values p < 0.05 considered statistically significant, (***p < 0.001).

Figure 3

CBD-X extract modulates TCR- signaling in human derived CD4⁺ T cells. CD4⁺ T cells were isolated from healthy donors by negative magnetic selection with the EasySep Direct Human CD4⁺ T cells Isolation Kit. Isolated CD4⁺ T cells were activated with 5 µg/ml anti-CD3 and 5 µg/ml anti-CD28 and treated with 1 or 2 µg/ml CBD-X extract or DMSO as a control. After an hour, the cells were collected and lysed with 100 µl 2x SB+βME. (A) Cell lysates were analyzed for the presence of phosphorylated STAT5, Lck and Zap70 by western blot analysis using pY694-STAT5, total STAT5, pY394-Lck, total Lck, pY319-Zap70 and total Zap70 antibodies. (B) Induction of phosphorylation was quantified relative to the specific total protein expression. Error bars represent the standard deviation of the means of three different donors, and they are expressed as average ± standard deviation (SD). SD were calculated in ratio to activated CD4 (treated with Anti-CD3/CD28), and data were analyzed by one-way ANOVA (Fisher’s LSD test with values p < 0.05 considered statistically significant, (*p <0.05, **p < 0.01, ***p < 0.001).

Figure 4

CBD-X extract inhibits the migration of T cells induced by SDF-1. PBMCs were isolated from the blood of healthy donors and leukocytes were isolated by the aid of the Lymphoprep-Density gradient medium. Leukocytes were activated by 2.5 µg/ml anti-CD3, 2.5 µg/ml anti-CD28 and 125 units/ml IL-2 for 48 hours. Activated PBMC derived T cells were seeded onto the 5 µm pore polyester membrane in the upper chamber of a 24-well Boyden chamber in RPMI 1640 medium. Human chemoattractant SDF-1 in a concentration of 100 nM was placed in the lower chamber. CBD-X extract in the concentrations of 1, 2 and 3 µg/ml or DMSO as a vehicle were added to the cells. After four hours, cells that have been migrated through the pores into the lower chamber were collected and stained with the Fc Receptor Blocking Solution Human TruStain FcX™ and anti PE- CD3, APC- CD4 and anti-Pacific blue-CD8 to distinguish T cell subtypes and subjected to flow cytometry analysis. (A) The number of migrated CD3⁺ T cells (B) of migrated CD4⁺ T cells and (C) of migrated CD8⁺ T cells were determined. The means were calculated from healthy donors (black line) and each dot represents one case. Data were analyzed in comparison to “SDF1, DMSO” group by and analyzed by One-way ANOVA (Fisher's LSD test with values p < 0.05 considered statistically significant, (*p < 0.05, **p < 0.01, ***p < 0.001).

Figure 5

CBD-X extract promotes immune reprogramming in a murine model of systemic inflammation. C57BL/6 mice were injected intravenously with 100- 150 mg/kg CBD-X extract or vehicle as a control. Simultaneously, 1 mg/kg LPS or PBS as a control were injected intraperitoneally. After two hours, mice were euthanized. Then, blood was drained from the heart and peritoneal contents were collected using PBS through lavage. Blood was centrifuged and supernatants were collected. Levels of TNF-α, IL-1β and IL-10 from the peritoneum (upper panel, A–C) or blood (lower panel, D–F) were detected by ELISA. Each colored dot represents one mouse. Standard deviations were calculated as the means of three biologically independent experiments (black line), each experiment included five mice per group. Data were analyzed by one-way ANOVA (Fisher’s LSD test with values p < 0.05 considered statistically significant, (*p < 0.05, **p < 0.01, ***p < 0.001).

Figure 6

CBD-X extract promotes immune silencing in a murine model of lung inflammation. C57BL/6 mice were injected intravenously with 100- 150 mg/kg CBD-X extract or vehicle as a control. Simultaneously, 1 mg/kg LPS or PBS as a control were administered intranasally. After two hours, mice were euthanized, and lung fluids were collected using PBS through lavage. The lung fluids were centrifuged, and supernatants were collected. Levels of the pro-inflammatory IL-1β (A), MCP-1 (B) and TNF-α (C) were detected by ELISA. Each colored dot represents one mouse. Standard deviations were calculated as the means of three biologically independent experiments (black line), each experiment included five mice per group. Data were analyzed by one-way ANOVA (Fisher’s LSD test with values p < 0.05 considered statistically significant, (*p <0.05, **p < 0.01, ***p < 0.001).

Figure 7

CBD-X extract inhibits migration of leukocytes to inflamed lungs LPS treated mice were injected intravenously with 150 mg/kg CBD-X extract or vehicle as a control. After 24 hours, mice were sacrificed, and lung fluids were collected. (A) Cells in the lung fluids were stained with TruStain FcX™ (anti-mouse CD16/32) and anti-mouse APC-CD45 for flow cytometry analysis. Vehicle-treated control cells are shown in the upper panel, LPS-treated cells in the middle panel and LPS and CBD-X treated cells in the lower panel. (B) Fold of change in APC-CD45 was calculated. Vehicle-treated control cells were set to 1 and LPS and LPS+CBD-X-treated cells were calculated to vehicle-treated control. Error bars represent the standard deviation of the mean of five mice/per group of three biologically independent experiments in ratio to control. (C) The cytokine IL-6 was detected by ELISA in the lung fluid. The means were calculated (black line) and each dot represents one mouse. Data were analyzed in comparison to “LPS, Vehicle” group by one-way ANOVA (Fisher's LSD test with values p < 0.05 considered statistically significant, (***p < 0.001). (D) Histological images of neutrophils infiltrated into the lungs of mice. Vehicle-treated neutrophils (left image), LPS-treated neutrophils (middle image) and LPS+CBD-X-treated neutrophils are shown. Representative images of one mice of each group of three different biologically independent experiments are shown at a magnification of X200.