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. 2015 Apr;172(7):1792-806.
doi: 10.1111/bph.13026. Epub 2015 Feb 10.

Δ(9) Tetrahydrocannabinol Attenuates Staphylococcal Enterotoxin B-induced Inflammatory Lung Injury and Prevents Mortality in Mice by Modulation of miR-17-92 Cluster and Induction of T-regulatory Cells

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Free PMC article

Δ(9) Tetrahydrocannabinol Attenuates Staphylococcal Enterotoxin B-induced Inflammatory Lung Injury and Prevents Mortality in Mice by Modulation of miR-17-92 Cluster and Induction of T-regulatory Cells

R Rao et al. Br J Pharmacol. .
Free PMC article

Abstract

Background and purpose: Staphylococcal enterotoxin B (SEB) is a potent activator of Vβ8+T-cells resulting in the clonal expansion of ∼30% of the T-cell pool. Consequently, this leads to the release of inflammatory cytokines, toxic shock, and eventually death. In the current study, we investigated if Δ(9) tetrahydrocannabinol (THC), a cannabinoid known for its anti-inflammatory properties, could prevent SEB-induced mortality and alleviate symptoms of toxic shock.

Experimental approach: We investigated the efficacy of THC against the dual administration (intranasal and i.p.) of SEB into C3H/HeJ mice based on the measurement of SEB-mediated clinical parameters, including cytokine production, cellular infiltration, vascular leak, and airway resistance. In addition, the molecular mechanism of action was elucidated in vitro by the activation of splenocytes with SEB.

Key results: Exposure to SEB resulted in acute mortality, while THC treatment led to 100% survival of mice. SEB induced the miRNA-17-92 cluster, specifically miRNA-18a, which targeted Pten (phosphatase and tensin homologue), an inhibitor of the PI3K/Akt signalling pathway, thereby suppressing T-regulatory cells. In contrast, THC treatment inhibited the individual miRNAs in the cluster, reversing the effects of SEB.

Conclusions and implications: We report, for the first time a role for the miRNA 17-92 cluster in SEB-mediated inflammation. Furthermore, our results suggest that THC is a potent anti-inflammatory compound that may serve as a novel therapeutic to suppress SEB-induced pulmonary inflammation by modulating critical miRNA involved in SEB-induced toxicity and death.

Figures

Figure 1
Figure 1
THC prevents mortality and alleviates SEB-induced inflammation in the lung. (A) Survival curve of mice receiving SEB+ vehicle when compared with mice treated with SEB+THC. (B) Measurement of airway hyperreactivity (sRAW) using whole-body plethysmography. (C) Assessment of vascular leak in the lungs; % vascular leak was calculated by measuring absorbance at 620 nm. (D) Histopathological examination of lungs as determined by haemotoxylin and eosin staining. Total layers of infiltrating cells were counted around 10 different capillaries and enumerated in the bar graph. (E) Total number of infiltrating mononuclear cells obtained from the lung was enumerated by trypan blue exclusion method. (F) Flow cytometric analysis to identify immune subsets was carried out. Mononuclear cells were stained with antibodies against T-cells (CD3), T-helper cells (CD4), cytotoxic T-cells (CD8), Vβ8-region of the T-cell receptor (Vβ8), NK cells and natural killer T-cells (NKT). Absolute cell numbers were calculated using the formula: total number of cells isolated from the lungs × % of specific cells/100, and plotted as a bar graph. Bar graphs summarize the means ±SEM from 3–5 independent experiments. All experiments above were carried out 72 h after exposure to SEB. Statistical significance is indicated as follows: *P < 0.005; **P < 0.01.
Figure 2
Figure 2
THC decreases SEB-induced cytokine secretion. (A) Measurement of early cytokines, IL-2 and MCP-1 in serum 3 h after SEB exposure. (B) Measurement of IFN-γ, IL-12, IL-10 and IL-6 in the BALF. All cytokine concentrations were determined using elisa. Bar graphs summarize the means ±SEM from 3–5 independent experiments. For cytokines from the serum, unpaired, two-tailed t-test was used to determine significance from SEB. For cytokines from the BALF, one-way anova, followed by post hoc analysis using Tukey's method was used.
Figure 3
Figure 3
THC significantly down-regulates SEB-induced expression of the miR-17-92 cluster. Real-time (RT) PCR validation of the individual miRNA (miR-17, miR-18a, miR-19a, miR-19b-1, miR20a and miR-92a-1) of the miR-17-92 cluster obtained from lung-infiltrating mononuclear cells. Data are normalized to internal control Snord_96a. Statistical significance was assessed using anova, Tukey's multiple comparison test.
Figure 4
Figure 4
The involvement of the miR-17-92 cluster in key biological pathways. (A) Graphical representation of the biological functions associated with significantly up-regulated SEB-induced miRNA as determined by IPA. (B) Canonical pathways associated with miRNA target genes. IPA was used to filter highly predicted and experimentally observed targets of only the significantly up-regulated miRNA in response to SEB. A graphical representation of the significant (Fisher's exact test) pathways of these particular target genes was generated. (C) IPA pathway demonstrating the convergence of the members of the miR-17-92 cluster on Pten.
Figure 5
Figure 5
miR-18a targets Pten, an inhibitor of the PI3K/Akt pathway. (A) RT-PCR of members of the miR-17-92 cluster in splenocytes that were activated with SEB and treated with AKT inhibitor (20 μM). (B) Target prediction of miR-18a using miRanda (www.microrna.org), showing the alignment of the mature miRNA to the 3′ UTR of Pten mRNA. The miRSVR score represents the probability of mRNA target down-regulation and the cut-off for a good score was set at ≤ −0.01. (C) RT-PCR quantification of miR-18a and its target Pten. Splenocytes were transfected with 40 nmol of a miR-18a mimic for 24 h. (D) RT-PCR quantification of miR-18a and Pten after transfection with a synthetic miR-18a inhibitor. Splenocytes that were activated with SEB (1 μg·mL−1) were also transfected with miR-18a inhibitor for 24 h. (E) Real-time PCR quantification of Pten levels in lung-infiltrating mononuclear cells. All miRNA levels were measured relative to internal control Snord_96a and mRNA levels were normalized to β-actin. Statistical significance was assessed using anova, Tukey's multiple comparison test.
Figure 6
Figure 6
THC is an inhibitor of the Akt pathway and leads to the induction of CD4+Foxp3+ T-regulatory cells. (A) IFN-γ levels in supernatants of splenocytes that were activated with SEB and treated with varying doses of THC (5 μM,10 μM and 20 μM) or Akt inhibitor (5 μM,10 μM or 20 μM) as indicated. (B) Thymidine incorporation to measure proliferation of splenocytes activated with SEB or treated with the indicated doses of THC (5 μM, 10 μM and 20 μM) or Akt inhibitor (5 μM,10 μM or 20 μM). (C) RT-PCR of Foxp3 levels in splenocytes that were treated either with THC (20 μM )or Akt inhibitor (20 μM). mRNA levels measured relative to β-actin. (D) Flow cytometric analysis of CD4+Foxp3+ T-regulatory cells in lung-infiltrating mononuclear cells for the groups indicated. The bar graph represents the RT-PCR expression of Foxp3 in lung-infiltrating mononuclear cells. In all in vitro experiments, splenocytes were activated with 1 μg·mL−1 SEB. Statistical significance was assessed using anova, Tukey's multiple comparison test.
Figure 7
Figure 7
Schematic of the proposed working model. SEB administration leads to activation of the PI3K/Akt pathway via the induction of the miR-17-92 cluster and the subsequent down-regulation of Pten. As a result, SEB exposure leads to increased cellular proliferation, cytokine production, pulmonary damage and acute mortality. The down-regulation of the cluster by THC restores Pten levels and allows for the inhibition of the PI3K/Akt axis. This leads to attenuation of acute inflammatory lung injury and induction of T-regulatory cells, which together prevent SEB-induced mortality.

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