The Endocannabinoid Anandamide Attenuates Acute Respiratory Distress Syndrome by Downregulating miRNA that Target Inflammatory Pathways

Abstract

Acute respiratory distress syndrome (ARDS) is defined as a type of respiratory failure that is caused by a variety of insults such as pneumonia, sepsis, trauma and certain viral infections. In this study, we investigated the effect of an endocannabinoid, anandamide (AEA), on ARDS induced in the mouse by Staphylococcus Enterotoxin B (SEB). Administration of a single intranasal dose of SEB in mice and treated with exogenous AEA at a dose of 40 mg/kg body weight led to the amelioration of ARDS in mice. Clinically, plethysmography results indicated that there was an improvement in lung function after AEA treatment accompanied by a decrease of inflammatory cell infiltrate. There was also a significant decrease in pro-inflammatory cytokines IL-2, TNF-α, and IFN-γ, and immune cells including CD4⁺ T cells, CD8⁺ T cells, Vβ8⁺ T cells, and NK⁺ T cells in the lungs. Concurrently, an increase in anti-inflammatory phenotypes such as CD11b + Gr1+ Myeloid-derived Suppressor Cells (MDSCs), CD4 + FOXP3 + Tregs, and CD4⁺IL10 + cells was observed in the lungs. Microarray data showed that AEA treatment in ARDS mice significantly altered numerous miRNA including downregulation of miRNA-23a-3p, which caused an upregulation of arginase (ARG1), which encodes for arginase, a marker for MDSCs, as well as TGF-β2, which induces Tregs. AEA also caused down-regulation of miRNA-34a-5p which led to induction of FoxP3, a master regulator of Tregs. Transfection of T cells using miRNA-23a-3p or miRNA-34a-5p mimics and inhibitors confirmed that these miRNAs targeted ARG1, TGFβ2 and FoxP3. In conclusion, the data obtained from this study suggests that endocannabinoids such as AEA can attenuate ARDS induced by SEB by suppressing inflammation through down-regulation of key miRNA that regulate immunosuppressive pathways involving the induction of MDSCs and Tregs.

Keywords: T regulatory cells (T regs); acute respiratory distress syndrome (ARDS); anandamide (AEA); miRNA-23a-3p; miRNA-34a-5p; micro-RNA (miRNA/miR); myeloid derived suppressor cells (MDSC); staphylococcus enterotoxin B (SEB).

FIGURE 1

AEA attenuates SEB-induced ALI in mice. Mice were exposed to SEB intra-nasally with a single dose of 50 µg/mouse on day 0. On days-1, 0 and 1, AEA or VEH was given into these mice i. p at a dose of 40 mg/kg body weight. Mice were euthanized on day 2 for various studies. (A) Showing clinical functions of the lung including Specific Airways Resistance (sRAW), Specific Airways Conductance (sGAW), and Minute per Volume (MV). (B) Comparison between the groups for the total number of Mononuclear Cells isolated from the lungs. (C) Representative images of histopathological H & E staining of excised lung tissue (20X magnification). (D) Measurement of cytokines IL2, IL6, and TNFα in the serum. Five mice in each group were used and the data confirmed in three independent experiments. *p ≤ 0. 05, **p ≤ 0. 01, ***p ≤ 0. 001.

FIGURE 2

AEA decreases T cell subpopulations in the lungs and spleens. Mice were treated with SEB and AEA as described in Figure 1 legend. Each panel shows a representative experiment depicting lung MNCs and splenocytes analyzed for various T cell markers. Data from five mice/group is presented in the form of vertical bars with Mean+/-SEM. Panels A–D and E-H show data from the lungs and spleens, respectively. (A,E) CD3⁺CD4⁺ T cells (B,F) CD3⁺CD8⁺ T cells, (C,G) CD3+Vβ8+ T cells, (D,H) CD3+NK1.1 + cells. Five mice in each group were used and the data was confirmed in three independent experiments. *p ≤ 0. 05, **p ≤ 0. 01, ***p ≤ 0. 001.

FIGURE 3

AEA decreases T cell subsets in the splenocytes activated with SEB in vitro. Spleen cells isolated from naïve mice were pretreated with AEA in vitro followed by activation with SEB, and then cultured for 24 (A-D) or 48 (E-H) hrs, and stained for various markers. Each panel shows a representative experiment using flow cytometry and the vertical bars depict data from groups of five mice with Mean+/-SEM. (A,E) CD3⁺CD4⁺ cells. (B, F) CD3⁺CD8⁺ cells. (C,G) CD3+Vβ8+ cells (D,H) CD3+NK + cells. The data was confirmed in three independent experiments. *p ≤ 0. 05, **p ≤ 0. 01.

FIGURE 4

AEA suppresses T cell activation markers in splenocytes activated with SEB. The spleen cells were pretreated with AEA and then activated with SEB in vitro for 48 h as described in Figure 3 legend. The cells were stained for various activation markers. Each panel shows a representative experiment using flow cytometry, and the vertical bars depict percentage data from groups of five mice with Mean+/-SEM. (A) CD3⁺CD69 ⁺ cells, (B) CD3⁺ cells, (C) CD69 + 25+ cells, (D) CD69 ⁺ cells, (E) CD44 ⁺ cells, (F) CD62L + cells. *p ≤ 0. 05, **p ≤ 0. 01, ***p ≤ 0. 001.

FIGURE 5

AEA alters miRNA expression in the mononuclear cells isolated from the lungs of SEB injected mice. Mice were treated with SEB and AEA as described in Figure 1 legend. Isolated mononuclear cells of the lung were screened for miRNA expression as described in methods (A). The heat map that shows all of the altered and unchanged miRNAs in the VEH + SEB and AEA + SEB groups. (B) Diagram of miRNAs with 1.5 fold change showing 59 miRNAs upregulated while 77 miRNAs downregulated. (C) Networking of the miRNAs with their targeted genes including anti-inflammatory genes, the map showing the main miRNAs including miRNA 23a-3p targeting ARG1 and TGFβ2 gene while miRNA 34a-5p targeting FOXP3 gene.

FIGURE 6

Validation of select miRNAs and targeted genes. Mice were treated with SEB and AEA as described in Figure 1 legend. Mononuclear cells from the lungs of both groups were isolated and screened for expression of miRNA expression with their targeted genes by qRT-PCR. (A) Binding affinity between miRNA 23a-3p and targeted genes including ARG1 and TGFβ2. (B) miRNA 23a-3p expression. (C,D) Expression of ARG1 and TGFβ2. (E) Binding affinity between miRNA 34a-5p and targeted gene FOXP3. (F) Expression of miRNA 34a-5p. (G) Expression FOXP3. Statistical significances as p ≤ 0. 05, **p ≤ 0. 01, ***p ≤ 0. 001.

FIGURE 7

Validation of the genes targeted by miRNA 23a-3p and miRNA 34a-5p. Splenocytes of C57BL6 mice, cultured and activated with SEB overnight were transfected with mock, mimic and inhibitor of each of miRNA 23a-3p and miRNA 34a-5p. qRT-PCR was used to detect the levels of targeted genes. (A) Expression of miRNA 23a-3p. (B) Expression of TGFB2 gene. (C) Expression of arginase one gene (D) Expression of miRNA 34a-5p. (E) Expression of FOXP3. Statistical significances as p ≤ 0. 05, **p ≤ 0. 01, ***p ≤ 0. 001.

FIGURE 8

AEA induces MDSCs and Tregs in the lungs of SEB administered mice. Mice were treated with SEB and AEA as described in Figure 1 legend. The lung MNCs were next stained for markers to detect MDSCs and Tregs. Each panel shows a representative experiment depicting lung MNCs analyzed for various T cell markers. Data from five mice/group is presented in the form of vertical bars with Mean+/-SEM. (A) Cells double-stained for CD11b and Gr1. a representative experiment using flow cytometry. (B) Cells double-stained for LY6C and LY6G. (C) Cells double-stained for CD4 and FOXP3. (D) Cells double-stained for CD4 and IL10. (E) Cells from the spleens were double-stained for CD11b and Gr1. Vertical bars show data from five mice. (F) AEA-induced MDSCs were incubated with splenic T cells that were activated with ConA at different ratios to create different Tcell:MDSC ratios. T cell proliferation was assessed by ³H- Thymidine Incorporation Assay. Data from five mice/group is presented in the form of vertical bars with Mean+/-SEM. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001.