Cannabis compounds exhibit anti-inflammatory activity in vitro in COVID-19-related inflammation in lung epithelial cells and pro-inflammatory activity in macrophages

Abstract

Cannabis sativa is widely used for medical purposes and has anti-inflammatory activity. This study intended to examine the anti-inflammatory activity of cannabis on immune response markers associated with coronavirus disease 2019 (COVID-19) inflammation. An extract fraction from C. sativa Arbel strain (F_CBD) substantially reduced (dose dependently) interleukin (IL)-6 and -8 levels in an alveolar epithelial (A549) cell line. F_CBD contained cannabidiol (CBD), cannabigerol (CBG) and tetrahydrocannabivarin (THCV), and multiple terpenes. Treatments with F_CBD and a F_CBD formulation using phytocannabinoid standards (F_CBD:std) reduced IL-6, IL-8, C-C Motif Chemokine Ligands (CCLs) 2 and 7, and angiotensin I converting enzyme 2 (ACE2) expression in the A549 cell line. Treatment with F_CBD induced macrophage (differentiated KG1 cell line) polarization and phagocytosis in vitro, and increased CD36 and type II receptor for the Fc region of IgG (FcγRII) expression. F_CBD treatment also substantially increased IL-6 and IL-8 expression in macrophages. F_CBD:std, while maintaining anti-inflammatory activity in alveolar epithelial cells, led to reduced phagocytosis and pro-inflammatory IL secretion in macrophages in comparison to F_CBD. The phytocannabinoid formulation may show superior activity versus the cannabis-derived fraction for reduction of lung inflammation, yet there is a need of caution proposing cannabis as treatment for COVID-19.

Figure 1

The levels of (a) IL-6 and (b) IL-8 in A549 cells treated with C. sativa Arbel crude and F_CBD and F_THC extract fractions. Cells were treated with 300 ng/mL TNFα and C. sativa extract and fractions at a concentration of 5 μg/mL for 4 h. Dexamethasone (Dex; 4 μg/mL) served as a positive control. Control (0.5% v/v methanol) treatment served as the solvent (vehicle) control; TNFα indicates TNFα+ solvent control treatment. Error bars indicate ± standard error (sem) (n = 3). Bars labeled with different letters are significantly different from all combinations of pairs by Tukey–Kramer honest significant difference test (HSD; P ≤ 0.05). Dose–effect curves of C. sativa F_CBD on (c) IL-6 and (d) IL-8 levels in the A549 cell line. Dose–effect curves of F_CBD:std (93.5% CBD+ 6.1% CBG+ 0.4% THCV) on (e) IL-6 and (f) IL-8 levels in the A549 cell line. GraphPad Prism version 6.1 was used to produce the dose–response curve and IC₅₀ doses. Error bars indicate ± sem (n = 3).

Figure 2

The level of (a) IL-6 and (b) IL-8 in A549 cells treated with F_CBD and CBD. Cells were treated with 300 ng/mL TNFα, 4.1 μg/mL F_CBD (FCBD) and CBD at different concentrations for 4 h. Dexamethasone (Dex; 4 μg/mL) served as a positive control. Control (0.6% v/v methanol) treatment served as the solvent (vehicle) control; TNFα is TNFα+ solvent control treatment. Error bars indicate ± sem (n = 3). Bars labeled with different letters are significantly different from all combinations of pairs according to the Tukey–Kramer HSD test (P ≤ 0.05).

Figure 3

Levels of (a) IL-6 and (b) IL-8 in A549 cells treated with F_CBD or F_CBD:std with or without CB1 or CB2 inverse agonists (IA), TRPA1 blocker, or TRPV1 or TRPV2 antagonists. Cells were treated with 300 ng/mL TNFα and F_CBD (FCBD) and F_CBD:std (FCBD:std) at a concentration of 3.4 and 4.1 μg/mL, respectively, in the presence or absence of IA of CB1 (5 µM) or CB2 (5 and 7.5 µM for IL-6 and IL-8, respectively), a TRPA1 blocker (10 µM), or TRPV1 or TRPV2 antagonists (10 µM) for 4 h. Dexamethasone (Dex) served as a positive control at 4 µg/mL. Control (0.4% v/v methanol + 2% v/v dimethyl sulfoxide [DMSO]) treatment served as the solvent (vehicle) control; TNFα is TNFα+ solvent control treatment. Error bars indicate ± sem (n = 3). Bars labeled with different letters are significantly different from all combinations of pairs according to the Tukey–Kramer HSD test (P ≤ 0.05).

Figure 4

qPCR-based determination of the mRNA steady state level in A549 cell line of (a) CCL2, (b) CCL7, (c) IL-7 or (d) ACE2 genes, after treatment with TNFα (300 µg/mL), F_CBD (FCBD; 7 µg/mL) or F_CBD:std (FCBD:std; 7 µg/mL), or Dexamethasone (Dex; 4 µg/mL)—for 6 h relative to the control. Control (0.7% v/v methanol) treatment served as the solvent (vehicle) control; TNFα indicates TNFα+ solvent control treatment. Error bars indicate ± sem (n = 3). Bars labeled with different letters are significantly different from all combinations of pairs according to the Tukey–Kramer HSD test (HSD; P ≤ 0.05).

Figure 5

qPCR-based determination of the mRNA steady state level in the differentiated KG1 cell line of (a) IL-6, (b) IL-8 or (c) CCL2 after treatment with TNFα (300 µg/mL), F_CBD (FCBD) at 7 µg/mL, F_CBD:std (FCBD:std) at 7 µg/mL and dexamethasone (Dex) at 8 µg/mL for 6 h relative to control. Control (0.7% v/v methanol) treatment served as the solvent (vehicle) control. (d) IL-8 levels in KG1 cells treated with F_CBD and F_CBD-std. Cells were treated with 300 ng/mL TNFα (and not by PMA), F_CBD (FCBD) or F_CBD:std (FCBD:std) at 10 μg/mL for 6 h. Dexamethasone (Dex; 4 μg/mL) served as a positive control. Control (1% v/v methanol) treatment served as the solvent (vehicle) control; TNFα is TNFα+ solvent control treatment. (e) IL-8 levels in KG1 cells treated with F_CBD. Cells were treated with 300 ng/mL TNFα (and not by PMA) and F_CBD at different concentrations for 6 h. Dexamethasone (Dex; 4 μg/mL) served as a positive control. Control (1.2% v/v methanol) treatment served as the solvent (vehicle) control; TNFα indicates TNFα+ solvent control treatment. Error bars indicate ± sem (n = 3). Bars with different letters are significantly different from all combinations of pairs according to the Tukey–Kramer HSD test (P ≤ 0.05).

Figure 6

Representative examples of confocal images of macrophages following treatment with the control and F_CBD (7 µg/mL). Control (0.7% v/v methanol) treatment served as the solvent (vehicle) control. Cell were stained for F-actin (EasyProbes™ ActinRed 555 Stain, red stain), and nuclei (Hoechst, blue stain); n = 5, in each biological replicate multiple cells were examined (see Table 2). Membrane filopodia-like structures are marked with white arrows. FNP, fluorescent-labeled silica 50–100 nm particles.

Figure 7

qPCR-based determination of the mRNA steady state level in the differentiated KG1 cell line. (a) FcγRII, (b) CD36 or (c) SCARB1 genes, after treatment with F_CBD (FCBD) at 7 µg/mL, F_CBD:std (FCBD:std) at 7 µg/mL, ruxolitinib (Ruxo) at 100 µg/mL or palmitic acid (PA) at 150 µM. In this experiment, controls (vehicle) for (a) and (c) were 0.7% v/v methanol + 2% v/v DMSO; for (b) 0.7% v/v methanol. Error bars indicate ± sem (n = 3). Bars labeled with different letters are significantly different from all combinations of pairs according to the Tukey–Kramer HSD test (P ≤ 0.05). *Indicates significantly different mean from the control based on the Student T-test (P ≤ 0.05).