In search of preventive strategies: novel high-CBD Cannabis sativa extracts modulate ACE2 expression in COVID-19 gateway tissues

Cannabis extracts regulate ACE2 expression in normal/unstimulated 3D tissues

Airway tissues

We analyzed the effect of cannabis extracts #5, #10, #31, #49, #81, #114, #155, #166, #169 and #207 on ACE2 expression in 3D EpiAirway tissues. Western blot analysis showed that extracts #5, #10, #31, #49, #114 and #155 significantly (p<0.05) down-regulated the ACE2 expression (Figure 2), whereas extracts #81 and #166 slightly increased its expression.

Figure 2. Effects of novel C. sativa extracts on the levels of ACE2 in the normal uninduced human EpiAirway tissue models. Two tissue samples were used per treatment group. Protein extracts were prepared from each sample, and equal amounts of each sample in each group were pooled together. Each bar is an average (with SD) from three technical repeat measurements. * - p<0.05, Student’s t-test.

Oral tissues

We also examined the effect of extracts #1, #7, #45, #317, #130 and #131 and of CBD alone on ACE2 expression in 3D EpiOral tissues. Western blot analysis indicated that extracts #1 and #7 down-regulated ACE2 expression (Figure 3), whereas CBD and extracts #45, #317 and #130 up-regulated its expression. These results suggest that cannabis extracts may differentially regulate ACE2 expression in normal human 3D tissues.

Figure 3. Effects of novel C. sativa extracts on the levels of ACE2 in the normal uninduced EpiOral tissue models. Three tissue samples were used per treatment group. Protein extracts were prepared from each sample, and equal amounts of each sample in each group were pooled together. Each bar is an average (with SD) from three technical repeat measurements. * - p<0.05, Student’s t-test.

Cannabis extracts modulate ACE2 expression in inflammation-stimulated 3D tissues

We examined the effect of cannabis extracts on the expression of ACE2 in inflammation-stimulated 3D tissues, since inflammation is a significant component of viral disease.

Oral tissues

The 3D EpiOral tissues were treated with TNFα/IFNγ for 24 h, followed by application of the indicated extracts for another 24 h. In an initial experiment, we used RNASeq to analyze the effects of four extracts (#81, #90, #130, #131) of the novel cannabis cultivars on the levels of ACE2 expression in the EpiOral tissues after inflammation induction by TNFα/IFNγ. Extracts #81 and #130 significantly down-regulated the levels of the ACE2 transcripts in the EpiOral tissues (ANOVA-like analysis, padj=2.14e-06 for both extracts; pair-wise comparison between DMSO and extract #130 and between DMSO and extract #81, padj<0.05) (Figure 4A). In a larger-scale follow-up experiment, we investigated the effect of nine extracts (#1, #7, #9, #45, #115, #129, #157, #167, #169) of the novel cannabis cultivars. The RNASeq analysis showed that TNFα/IFNγ up-regulated the ACE2 mRNA levels (Figure 4B), and all extracts except #115 down-regulated the expression of the TNFα/IFNγ-induced changes in ACE2, although not significantly. Interestingly, Western blot analysis showed that extracts #9 and #45 increased ACE2 protein levels (Figure 4C), whereas #1, #7, #115, #157, #167 and #169 down-regulated the protein levels.

Figure 4. Effects of novel C. sativa extracts on the levels of ACE2 in the EpiOral tissue models upon induction of inflammation by treatment with TNFα/IFNγ. (A, B) Total RNA was isolated from 3D EpiOral tissues and subjected to RNA-Seq analysis as described in the “Methods”. The levels of ACE2 gene expression is presented as an average (with SD) from two samples. * - Statistically significant, ANOVA-like analysis and pair-wise comparison, as per Materials and Methods. (C) Two tissue samples were used per treatment group. Whole lysates prepared from EpiOral tissues were subjected to Western blotting using antibody against ACE2 as described in the “Methods”. The relative densitometry is presented as an average (with SD) from three technical repeat measurements. * - p<0.05, Student’s t-test.

Intestinal tissues

We treated 3D EpiIntestinal tissues with TNFα/IFNγ for 24 h and then exposed the tissues to the indicated extracts for another 24 h. RNASeq analysis indicated that extract #45 significantly down-regulated the ACE2 mRNA levels (Figure 5A); extracts #129 and #130 had a tendency to decrease ACE2 expression, but the difference was not significant.

Figure 5. Effects of novel C. sativa extracts on the levels of ACE2 in the EpiIntestinal tissue models upon induction of inflammation by treatment with TNFα/IFNγ. (A) Total RNA was isolated from 3D EpiItestinal tissues and subjected to RNA-Seq analysis as described in the “Methods”. The levels of ACE2 gene expression is presented as an average (with SD) from two samples. * - Statistically significant, ANOVA-like analysis and pair-wise comparison, as per Materials and Methods. (B) Two tissue samples were used per treatment group. Whole lysates prepared from EpiIntestinal tissues were subjected to Western blotting using antibody against ACE2 as described in the “Methods”. The relative densitometry is presented as an average (with SD) from three technical repeat measurements. * - p<0.05, Student’s t-test.

Western blot analysis showed that, in addition to extracts #45 and #129, which decreased the ACE2 protein levels (Figure 5B) and confirmed the RNASeq results, extracts #1, #7 and #169 down-regulated the ACE2 protein levels, while #9, #115, #130 and #274 had no effect.

Airway tissues

To analyze the effects of the cannabis extracts on the ACE2 expression in the inflammation-stimulated airway tissues, we used two types of 3D tissue models: EpiAirway and EpiAirway Full Thickness (EpiAirway-FT). The full-thickness tissues contain mucociliary epithelium as well as stromal fibroblasts, unlike the EpiAirway tissues, which contain only the epithelial component (Figure 1).

To delineate the effects of the extracts on the inflammation-simulated EpiAirway-FT, we first treated the EpiAirway-FT tissues with either TNFα/IFNγ, or TNFα/IFNγ supplemented with single cannabinoids (CBD or cannabinol [CBN]) or with the indicated C. sativa extracts for 48 h. CBD was chosen because of its documented anti-inflammatory properties and its abundance in the studied extracts. CBN was selected as it is a non-psychoactive cannabinoid that has many properties similar to delta-9-tetrahydrocannabinol (Δ⁹-THC). Western immunoblotting showed that TNFα/IFNγ triggered an increase in the ACE2 expression in the EpiAirway-FT tissues (Figure 6A). Remarkably, all the examined extracts attenuated the TNFα/IFNγ-induced ACE2 expression, while CBD and CBN had no effect on ACE2 expression (Figure 6A). Similar results were also observed in an independent experimental repeat using the 3D EpiAirway tissues (Figure 6B). Together, these results suggest that the cannabis extracts modulated the ACE2 expression in the inflammation-stimulated 3D tissues, while CBD or CBN alone did not affect the ACE2 levels.

Figure 6. Effects of novel C. sativa extracts and cannabinoids on the levels of ACE2 in the EpiAirway-FT (A) and EpiAirway (B) tissue models upon induction of inflammation by treatment with TNFα/IFNγ. Three tissue samples were used per treatment group. Protein extracts were prepared from each sample, and equal amounts of each sample in each group were pooled together. Each bar is an average (with SD) from three technical repeat measurements. * - p<0.05, Student’s t-test.

Cannabis extracts affect TMPRSS2 expression

Along with ACE2, the serine protease TMPRSS2 plays an important role in the SARS-CoV-2 infection process. While ACE2 is the receptor for viral entry, TMPRSS2 primes viral spike proteins and is therefore crucial for SARS-CoV-2 entry into host cells. Recent studies reveal that TMPRSS2 inhibitors block virus entry [17]. RNASeq analysis indicated that TNFα/IFNγ triggered TMPRSS2 expression, which was somewhat attenuated by all the extracts tested (Figure 7).

Figure 7. Effects of novel C. sativa extracts on the levels of TMPRSS2 gene expression in the EpiOral and EpiIntestinal tissue models. Each bar is an average (with SD) of two samples, as per Materials and Methods. The Y axis shows the arbitrary units of TMPRSS2 gene expression, while the X axis shows the samples.

We further explored whether or not the cannabis extracts affected the TMPRSS2 protein levels in the oral and airway tissues. We found that application of extracts #1, #45, #317 and #131 decreased TMPRSS2 levels compared with CBD alone, which up-regulated TMPRSS2 in the oral tissues (Figure 8). Tested extracts (except #131) and CBD increased the TMPRSS2 levels compared with DMSO.

Figure 8. Effects of novel C. sativa extracts on the levels of TMPRSS2 in the normal uninduced EpiOral tissue models. Three tissue samples were used per treatment group. Protein extracts were prepared from each sample, and equal amounts of each sample in each group were pooled together. Each bar is an average (with SD) from three technical repeat measurements. * - p<0.05, Student’s t-test.

In the TNFα/IFNγ-stimulated EpiAirway-FT tissues, TNFα/IFNγ increased the TMPRSS2 levels. Application of CBD, CBN and extracts #81 and #129 increased the TMPRSS2 protein levels, while extracts #5, #10 and #317 slightly decreased them compared with TNFα/IFNγ alone (Figure 9A). In the TNFα/IFNγ-stimulated EpiAirway tissues, extracts #1, # 5, #7 and #45 down-regulated the TNF/IFN-induced TMPRSS2 expression, while extracts #10 and #129, CBD and CBN did not affect its levels (Figure 9B).

Figure 9. Effects of novel C. sativa extracts on the levels of TMPRSS2 in the EpiAirway-FT (A) and EpiAirway (B) tissue models upon induction of inflammation by treatment with TNFα/IFNγ. Three tissue samples were used per treatment group. Protein extracts were prepared from each sample, and equal amounts of each sample in each group were pooled together. Each bar is an average (with SD) from three technical repeat measurements. * - p<0.05, Student’s t-test.

To determine whether ACE2 and TMPRSS2 inhibition was dependent on the level of a single cannabinoid, total cannabinoids or their ratio, we correlated the down-regulation level of ACE2 and TMPRSS2 with the level of cannabinoids for each experiment. Interestingly, we found a negative correlation between the down-regulation fold of ACE2 and all individual cannabinoids or total cannabinoid content in most experiments (Supplementary Table 1).

Next, we analyzed the correlation between ACE2 and TMPRSS2 effects and total terpene concentrations for several cultivars for which we had data (extracts #1, #7, #9, #81 and #317) (Table 2). We found a strong positive correlation between the efficiency of the extracts in the down-regulation of ACE2 and the total level of terpenes in these extracts in experiments analyzing the EpiOral, EpiIntenstinal and EpiAirway transcriptomes (0.99, 0.97 and 0.97, respectively, as seen in Supplementary Table 2). In contrast, the correlation between the terpenes and protein levels (Western blot) was not as evident; weak negative correlations were found for the EpiOral and EpiIntestinal tissues (−0.31 and −0.29, respectively). The correlation for TMPRSS2 was also weak.

We next compared the degree of down-regulation of ACE2 in the different tissues and cells. We found that, on average, the effect was most prominent on the EpiAirway tissues. As far as the individual extracts were concerned, the strongest effect was observed for extracts #1, #129, #7 and #5 (Supplementary Table 3).

Plant growth, extract preparation

All cannabis plants were grown in the licensed facility at the University of Lethbridge (license number LIC-62AHHG0R77-2019). C. sativa cultivars #1, #5, #7, #9, #10, #31, #45, #49, #81, #90, #114, #115, #129, #130, #131, #155, #157, #166, #167, #169, #207, #274, #317 were used for the experiments. Four plants per cultivar were grown at 22° C 18 h light 6 h dark for 4 weeks and then transferred to the chambers with 12 h light/12 h dark regime to promote flowering. Plants were grown to maturity and flowers were harvested and dried. Flower samples from four plants per cultivar were combined and used for extraction. Three grams of the powdered plant tissue per each line were used for extraction. Plant material was placed inside a 250 mL Erlenmeyer flask, 100 mL of Ethyl Acetate was poured into each flask. The flasks were covered with tin foil and incubated overnight in the dark at 21C with continuous shaking at 120 rpm. Extracts were filtered, concentrated using a rotary vacuum evaporator and transferred to a tared 3-dram vial. The leftover solvent was evaporated to dryness in an oven overnight at 50° C to eliminate the solvent completely. Levels of cannabinoids was analysed using Agilent Technologies 1200 Series HPLC system. The extract stocks were prepared from the crude extracts whereby 3-6 mg of crude extract were dissolved in DMSO (Dimethyl sulfoxide anhydrous, Life Technologies) to reach 60 mg/mL final concentration and stored at -20° C. Appropriate cell culture media (RPMI + 10% FBS or EMEM + 10% FBS) were used to dilute the 60 mg/mL stock to make working medium containing 0.01 mg/mL. Extracts were sterilized using 0.22 μm filter. Extract composition are shown in Table 1.

Tissue models and treatments

Tissue models

EpiAirway^TM, EpiAirwayFT^TM, EpiOral^TM, EpiIntestinal^TM tissues were purchased from Mattek Life Sciences (Ashland, MA), equilibrated for 24 h and cultured according to manufacturer’s instructions.

EpiAirway tissues (AIR-100)

Mattek’s EpiAirway tissue model is a human 3D mucociliary tissue model that consists of normal, human-derived tracheal/bronchial epithelial cells, is cultured at the air-liquid interface and fully recapitulates the barrier, mucociliary responses, infection, toxicity responses of human airway tissues in vivo (Mattek Life Sciences, MA). Two tissues were used per extract in experiments shown in Figure 3. Three tissues were used per extract in experiments shown in Figures 6B, 9B.

EpiOral tissues (ORL-200)

MatTek’s EpiOral tissues consist of normal, human-derived oral epithelial cells. The cells have been cultured to form multilayered, highly differentiated models of the human buccal (EpiOral) phenotypes. The tissues are cultured on specially prepared cell culture inserts using serum free medium and attain levels of differentiation on the cutting edge of in vitro cell culture technology. The EpiOral tissue models exhibit in vivo-like morphological and growth characteristics which are uniform and highly reproducible (Mattek Life Sciences, MA). Two tissues were used per extract in experiments shown in Figures 4, 7. Three tissues were used per extract in experiments shown in Figures 3, 8.

EpiIntestinal tissues (SMI-100)

EpiIntestinal tissues are 3D highly differentiated tissue models produced from normal, human cell-derived small intestine epithelial and endothelial cells and fibroblasts. Grown at the air-liquid interface, EpiIntestinal tissue models are similar to in vivo human epithelial tissues and exhibit columnar shaped basal cells and Kerckring folds, as well as brush borders, functional tight junctions and mucous secreting granules (Mattek Life Sciences, MA). Two tissues were used per extract in experiments shown in Figures 5, 7.

EpiAirwayFT (AFT-100)

EpiAirwayFT is a ready-to-use, 3D mucociliary tissue model consisting of normal, human-derived tracheal/bronchial epithelial cells and normal human stromal fibroblasts (Mattek Life Sciences, MA). H&E-stained section of EpiAirwayFT tissue (courtesy of Mattek) exhibits a pseudostratified epithelium with ciliated cells and an extracellular matrix containing fibroblasts on a microporous membrane (Figure 1). Three tissues were used per extract (Figures 6A, 9A).

Treatments

EpiAirway tissue treatment (Figure 2)

Tissues were equilibrated for 24 h. The extracts or vehicle (DMSO) were dissolved in media and applied to the media surrounding the tissues. Tissues were incubated with 0.015 μg/μL of extracts for 24 h and flash frozen for RNA and protein analysis. Two tissues were used per treatment.

EpiOral tissue treatment (Figures 3, 8)

Tissues were equilibrated for 24 h, after equilibration tissues were treated with 10 μM CBD or 0.015 μg/μL extracts for 24 h. Three tissues were used per treatment.

EpiIntestinal and EpiOral tissue treatments (data shown in Figures 4, 5)

Tissues were equilibrated for 24 h. Inflammation was induced by treatment with proinflammatory cytokines (10 ng/ml TNFα /IFN γ for 24 hours) and then the extracts or vehicle (DMSO) were dissolved in media and applied to the media surrounding the tissues. Tissues were further incubated with 0.015 μg/μL of extracts for 24 h and flash frozen for RNA and protein analysis. Two tissue were used per treatment.

EpiAirway and EpiAirway FT tissue treatments (Figures 6, 7, 9)

Tissues were equilibrated for 24 h, after equilibration tissues were treated with 10 ng/mL TNFα /IFN γ alone or in combination with 5 μM CBD, 5 μM CBN, or 0.015 μg/μL extracts for 48 h. Three tissues were used per treatment.

Gene expression analysis

RNA extraction

Two tissues per group were used for the analysis of gene expression profiles. RNA was extracted from tissues using TRIzol® Reagent (Invitrogen, Carlsbad, CA), further purified using an RNAesy kit (Qiagen), and quantified using Nanodrop2000c (ThermoScientific). Afterwards, RNA integrity and concentration were established using 2100 BioAnalyzer (Agilent).

Library construction and sequencing

In all cases, the sequencing libraries were prepared using NEBNext Ultra II mRNA library preparation kit for Illumina (NEB) following the manufacturer’s instructions. The samples were processed by the same technician at the same time to avoid the introduction of technical batch effects. The cDNA fragment libraries were sequenced using NextSeq500 sequencing analyzer (Illumina). The samples were balanced evenly across the lanes of the sequencing flowcell.

Statistics and bioinformatics analysis

Base-calling and demultiplexing were done with Illumina CASAVA v.1.9 bioinformatics pipeline. The base qualities were examined using FastQC v.0.11.8. The adapters and low-quality bases were trimmed using Trim Galore! v.0.6.4 www.bioinformatics.babraham.ac.uk/projects/trim_galore/. Trimmed reads were mapped to the human genome version GRCh37 using HISAT2 version 2.0.5 [31]. Counts of reads mapping to the gene as a meta-feature were obtained using featureCounts v.1.6.1 [32] taking to account the directionality of the sequencing libraries. Counts of reads mapping to features were loaded into R v.3.6.1 and normalized using DESeq2 v.1.24.0 Bioconductor package as described in the manual [33].

Two samples were used per group. The differences between all experimental groups were examined using the likelihood ratio test (LRT) test implemented in DESeq2. The reduced model included the intercept and the full model was the experimental group (Cannabis extracts and controls). Multiple comparisons adjustment of p-values was conducted by Benjamini-Hochberg procedure [34]. Specific comparisons between groups were extracted using results() function with contrast argument specified. Genes with adjusted p-values below 0.05 were considered significant. The results of statistical tests for the ACE2 receptor (Ensembl gene identifier: ENSG00000130234) were selected from the list of significant genes.

Western blot analysis

After treatment with cannabis extracts for the indicated time, whole cellular lysates of 3D tissues were prepared in radioimmunoprecipitation assay buffer using 2.0 mm ZR BashingBead beads (Zymo Research). tissue lysates were prepared from all samples, then equal amount of each sample from each group were pooled together. Proteins (30-100 μg per sample) were electrophoresed in 10% sodium dodecyl sulfate polyacrylamide gel and electrophoretically transferred to polyvinylidene difluoride membranes (Amersham Hybond^TM-P, GE Healthcare) at 4° C for 1.5 h. The blots were incubated for 1 h with 5% nonfat dry milk to block nonspecific binding sites and subsequently incubated at 4° C overnight with 1:1000 dilution of polyclonal antibody against ACE2 (Abcam) or TMPRSS2 (Abcam). Immunoreactivity was detected using a peroxidase-conjugated antibody and visualized with the ECL Plus Western Blotting Detection System (GE Healthcare). The blots were stripped before reprobing with antibody against GAPDH (Santa Cruz Biotechnology). Quantification of Western blot bands was performed using ImageJ in duplicate or triplicate as technical replicates.

Statistics

The student’s t test was used to determine the statistical significance of differences between groups in ACE2 and TMPRSS2 expression. A value of p < 0.05 was considered statistically significant.