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. 2020 Aug 28;21(17):6244.
doi: 10.3390/ijms21176244.

Δ9-Tetrahydrocannabinol Prevents Mortality from Acute Respiratory Distress Syndrome through the Induction of Apoptosis in Immune Cells, Leading to Cytokine Storm Suppression

Affiliations

Δ9-Tetrahydrocannabinol Prevents Mortality from Acute Respiratory Distress Syndrome through the Induction of Apoptosis in Immune Cells, Leading to Cytokine Storm Suppression

Amira Mohammed et al. Int J Mol Sci. .

Abstract

Acute Respiratory Distress Syndrome (ARDS) causes up to 40% mortality in humans and is difficult to treat. ARDS is also one of the major triggers of mortality associated with coronavirus-induced disease (COVID-19). We used a mouse model of ARDS induced by Staphylococcal enterotoxin B (SEB), which triggers 100% mortality, to investigate the mechanisms through which Δ9-tetrahydrocannabinol (THC) attenuates ARDS. SEB was used to trigger ARDS in C3H mice. These mice were treated with THC and analyzed for survival, ARDS, cytokine storm, and metabolome. Additionally, cells isolated from the lungs were used to perform single-cell RNA sequencing and transcriptome analysis. A database analysis of human COVID-19 patients was also performed to compare the signaling pathways with SEB-mediated ARDS. The treatment of SEB-mediated ARDS mice with THC led to a 100% survival, decreased lung inflammation, and the suppression of cytokine storm. This was associated with immune cell apoptosis involving the mitochondrial pathway, as suggested by single-cell RNA sequencing. A transcriptomic analysis of immune cells from the lungs revealed an increase in mitochondrial respiratory chain enzymes following THC treatment. In addition, metabolomic analysis revealed elevated serum concentrations of amino acids, lysine, n-acetyl methionine, carnitine, and propionyl L-carnitine in THC-treated mice. THC caused the downregulation of miR-185, which correlated with an increase in the pro-apoptotic gene targets. Interestingly, the gene expression datasets from the bronchoalveolar lavage fluid (BALF) of human COVID-19 patients showed some similarities between cytokine and apoptotic genes with SEB-induced ARDS. Collectively, this study suggests that the activation of cannabinoid receptors may serve as a therapeutic modality to treat ARDS associated with COVID-19.

Keywords: acute respiratory distress syndrome; apoptosis; cytokine storm; staphylococcal enterotoxin B; Δ9-tetrahydrocannabinol.

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Conflict of interest statement

The authors declare that they have no conflict of interest.

Figures

Figure 1
Figure 1
Δ9-Tetrahydrocannabinol(THC) attenuates staphylococcal enterotoxin-B, SEB-induced acute respiratory distress syndrome (ARDS) in mice. For in vivo studies, SEB-mediated ARDS was induced in C3H/HeJ mice, then the mice were treated with either vehicle (Veh) or THC, as described in Methods. For in vitro studies, epithelial cell type II was cultured, and the resistance was measured after adding splenocytes, which were activated with SEB + Veh or SEB+THC. (A): Representative hematoxylin and eosin, H&E images of lung tissue sections. (B): ECIS measurement of epithelial resistance, presented resistance is normalized to a pre-treatment time point for comparison of THC effect on barrier function. (C,D): ELISA quantification of broncho-alveolar lavage fluid (BALF) for cytokines (C) and chemokines (D). Vertical bars show data from 5 mice with mean+/-SEM. Statistical significance is depicted as * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001 between the groups.
Figure 2
Figure 2
THC attenuates SEB-induced acute respiratory distress syndrome (ARDS) in mice via the induction of apoptosis. For in vivo studies, SEB-mediated ARDS was induced in C3H/HeJ mice, then the mice were treated with either Veh or THC, as described in Methods. For in vitro studies, splenocytes were isolated from naïve mice and activated in culture with SEB+Veh or SEB+THC. (A): Terminal deoxynucleotidyl transferase dUTP nick end labeling, TUNEL staining of lung mono-nuclear cells MNCs isolated from SEB+Veh and SEB+THC mice. (B): DiOC6(3) staining of CD3+ MNCs isolated from SEB+Veh and SEB+THC mice. (C): DiOC6(3) staining of CD3+ splenocytes that were activated in vitro with SEB+Veh or SEB+THC (10 uM). (D): 3H- Thymidine incorporation assay of splenocytes activated in vitro for 72 h with SEB (1µg/mL) +Veh or SEB+THC (5µm or 10µm). Thymidine incorporation is shown as counts per minute (CPM). (E): Heatmap of genes associated with apoptosis in SEB+VEH vs. SEB+THC lung MNCs. Vertical bars in panels A-C show data from 5 mice with mean+/-SEM. Statistical significance is depicted as * p < 0.05 and **** p < 0.0001 between the groups.
Figure 3
Figure 3
Single-cell RNA sequencing of the lungs reveals that THC upregulates genes associated with apoptosis in SEB-challenged mice. Mice were treated with SEB+Veh or SEB+THC, as described in the Figure 1 legend. (A): scRNA-seq t-Distributed Stochastic Neighbor Embedding (tSNE) colored by cell type. (B): scRNA-seq tSNE split by sample ID and colored by cell type. (C): Violin plots of Bad expression amongst the clusters in SEB+Veh vs. SEB+THC. (D): Violin plots of Bax expression amongst the clusters in SEB+Veh vs. SEB+THC. (E): Violin plots of the Cox4i1c expression amongst the clusters in SEB+Veh vs. SEB+THC. (F): Violin plots of the Apopt1 expression amongst the clusters in SEB+Veh vs. SEB+THC. (G): Violin plots of the Casp3 expression amongst the clusters in SEB+Veh vs. SEB+THC. (H): Violin plots of Slc25a3 and Slc25a39 in CD8+ and CD4+ T cells.
Figure 4
Figure 4
THC treatment decreases T cell activation and alters metabolism to induce apoptosis. Spleen cells were cultured with SEB+THC or SEB+Veh for 72 h, followed by the purification of CD3+ T cells, as described in Methods. (A): OCR in cells during the mitochondrial stress test. (B): OCR in the glucose oxidation dependency test. (C): OCR in the β-oxidation dependency test. (D): Heat map showing dysregulated apoptosis-related metabolites from metabolome analysis of serum (n = 4). (EH): Concentrations of metabolites in serum from SEB+Veh and SEB+THC mice. (I): DiOC6(3) staining of splenocytes from C3H/HeJ mice activated with SEB (1 μg/mL) in the presence of either vehicle or PLC 200 μM for 72 h. (J): T cell proliferation measured by 3-H thymidine incorporation assay in splenocytes from C3H/HeJ mice activated with SEB (1 µg/mL) in the presence of either vehicle or PLC 200 µm for 72 h. (CPM, counts per minute). Statistical significances are depicted as * p < 0.05, ** p < 0.01, and **** p < 0.0001 between the groups.
Figure 5
Figure 5
THC treatment in SEB-injected mice results in altered miR expression in lung-infiltrating MNCs. Mice were treated with SEB+Veh or SEB+THC, as described in the Figure 1 legend. MNCs from the lungs were isolated and screened for miR expression. (A): IPA pathways and relationships between miRs and genes in lung MNCs. (B): qRT-PCR validation of miR-185-3p in lung-infiltrating MNCs. (C): Seed sequence alignments between miR-185-3p and Bad, Hrk, Runx3, Cox4 and Nkiras2. (DI) qRT-PCR validation of miR-185-3p targeted genes Bad (D), Bax (E), Hrk (F), Runx3 (G), Cox4 (H), and Nkiras2 (I) in lung-infiltrating MNCs. Statistical significance is depicted as * p < 0.05, *** p < 0.001, **** p < 0.0001 between the compared groups.
Figure 6
Figure 6
Comparison of SEB-induced cytokines and apoptosis from ARDS mice and COVID19 patients. (A): Venn diagram was used in the display of up-regulated genes related to proinflammatory cytokines from SEB-induced ARDS mice and COVID19 BALF patients. (B): Venn diagram was used for displaying common down-regulated genes related to the apoptosis pathway from SEB-induced ARDS mice and COVID19 BALF patients.

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