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. 2018 May;22(5):2644-2655.
doi: 10.1111/jcmm.13542. Epub 2018 Mar 7.

Resveratrol Protects Mice Against SEB-induced Acute Lung Injury and Mortality by miR-193a Modulation That Targets TGF-β Signalling

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Free PMC article

Resveratrol Protects Mice Against SEB-induced Acute Lung Injury and Mortality by miR-193a Modulation That Targets TGF-β Signalling

Hasan Alghetaa et al. J Cell Mol Med. .
Free PMC article

Abstract

Staphylococcal enterotoxin B (SEB) is a potent superantigen produced by Staphylococcus aureus that triggers a strong immune response, characterized by cytokine storm, multi-organ failure, and often death. When inhaled, SEB can cause acute lung injury (ALI) and respiratory failure. In this study, we investigated the effect of resveratrol (RES), a phytoallexin, on SEB-driven ALI and mortality in mice. We used a dual-exposure model of SEB in C3H/HeJ mice, which caused 100% mortality within the first 5 days of exposure, and treatment with RES resulted in 100% survival of these mice up to 10 days post-SEB exposure. RES reduced the inflammatory cytokines in the serum and lungs, as well as T cell infiltration into the lungs caused by SEB. Treatment with RES also caused increased production of transforming growth factor-beta (TGF-β) in the blood and lungs. RES altered the miRNA profile in the immune cells isolated from the lungs. Of these, miR-193a was strongly induced by SEB and was down-regulated by RES treatment. Furthermore, transfection studies and pathway analyses revealed that miR-193a targeted several molecules involved in TGF-β signalling (TGFβ2, TGFβR3) and activation of apoptotic pathways death receptor-6 (DR6). Together, our studies suggest that RES can effectively neutralize SEB-mediated lung injury and mortality through potential regulation of miRNA that promote anti-inflammatory activities.

Keywords: 3,4,5-trihydroxy-trans-stilbene; acute lung injury; death receptor 6; miR-193; microRNA (miRNA/miR); pulmonary disease; resveratrol; staphylococcal enterotoxin B; transforming growth factor-beta.

Figures

Figure 1
Figure 1
Effect of RES on clinical parameters of SEB‐induced ALI. Mice were given two doses of SEB as described in Methods. The first dose of RES was given 24 h prior to SEB exposure (day‐1) and the second at 90 min (Day 0) before the second challenge with SEB. The mice were studied for the next 10 d. (A) Survival curve over 10 d for SEB + VEH (n = 5) and SEB + RES (n = 5) groups. (B) Concentration of Evans blue dye in isolated lungs was used to study vascular permeability. (C) Representative histopathological H&E slides of excised lungs; (D) Pulmonary function tests depicting specific airway resistance (sRaw). Significance was measured in panel D only as following: √ = P < .01, √√ = P < .0001 in comparison between Naïve and SEB + VEH or SEB + RES groups. α = P < .05, β = P < .01, χ = P < .001 in comparison between SEB + VEH and SEB + RES groups in panel D. Significance in panels A and B was depicted as follows: *P < .05, **P < .01
Figure 2
Figure 2
Resveratrol (RES) decreases cellular infiltration in the lungs of SEB‐treated mice. Mice were exposed to SEB and treated with RES as described in Figure 1 legend. (A) Total number of mononuclear cells isolated from the lungs. (B) Total number of T cell subsets analysed after antibody staining and flow cytometry analysis. Significance was depicted as follows: *P < .05, **P < .01, ***P < .001
Figure 3
Figure 3
Effect of RES on serum cytokines induced by SEB. Mice were treated with SEB and RES as described in Figure 1 legend. Serum was collected from all experimental groups (Naïve, SEB + VEH, SEB + RES) 3 h after second dose of SEB. ELISA kits were used to detect (A) TNFα, (B) IL‐2, (C) MCP‐1, (D) IFN‐γ, (E) IL‐6 and (F) TGF‐β. Significance was depicted as follows: *P < .05, **P < .01, ***P < .001, ****P < .0001
Figure 4
Figure 4
Effect of RES on SEB‐induced serum cytokine levels at 48 h. Serum was collected from all experimental groups (Naïve, SEB + VEH, SEB + RES) 48 h after second dose of SEB. ELISA kits were used to detect (A) TNFα, (B) IL‐2, (C) MCP‐1, (D) IFN‐γ, (E) IL‐6 and (F) TGF‐β. Significance was depicted as follows: *P < .05, **P < .01, ***P < .001, ****P < .0001
Figure 5
Figure 5
Effect of RES on SEB‐induced BALF cytokine levels at 48 h. BALF was collected from all experimental groups (Naïve, SEB + VEH, SEB + RES) 48 h after second dose of SEB. ELISA kits were used to detect (A) TNFα, (B) IL‐2, (C) MCP‐1, (D) IFN‐γ, (E) IL‐6 and (F) TGF‐β. Significance was depicted as follows: *P < .05, **P < .01, ***P < .001, ****P < .0001
Figure 6
Figure 6
Resveratrol (RES) alters miRNA expression profiles in mononuclear cells isolated from SEB‐exposed mice. Mononuclear cells were isolated from the lungs of mice treated with SEB + VEH or SEB + RES as described in Figure 1 legend, and miRNA array was performed using GeneChip miRNA 4.0 platform. (A) Heatmap comparing SEB + VEH and SEB + RES groups with miRNA expression profiles (green indicates less intensity and red indicates an increase). (B) Venn diagram depicting fold changes of 3137 detected miRs that were either up (red), down (green) or not changed (yellow) significantly when comparing SEB + VEH and SEB + RES groups. Significant changes were determined to be greater or lesser than 2 linear fold change. (C) IPA‐derived network depicting the relationship of altered miR to their potential gene targets. (D) IPA‐derived network depicting the relationship of miR‐193a to altered signalling and regulatory pathways
Figure 7
Figure 7
Validation of miR‐193a and its targets. (A) Gene target alignment scores (mirSVR and PhastCons) from microrna.org are depicted for miR‐193a‐3p and gene targets TGFB2, TGFBR3 and DR6. (B) miR‐193a expression was validated with qRT‐PCR from RNA samples collected from lung mononuclear cells from naïve, SEB + VEH and SEB + RES groups. Data are expressed as fold change compared to naïve controls. (C, D, E and F) qRT‐PCR expression levels of TGF‐β1, TGFβ2, TGFβR3 and DR6, respectively, in infiltrated mononuclear cells into lungs of SEB + VEH and SEB + RES groups compared to naïve controls. (G) Validation qRT‐PCR for the expression of miR‐193a‐3p in SEB‐activated splenocytes when transfected with mock, mimic or inhibitor for miR‐193a‐3p. Data are expressed as fold change compared to mock controls. (H, I, and J), qRT‐PCR values from TGFB2, TGFBR3 and DR6 after transfection of SEB‐activated splenocytes with either mock, mimic or inhibitor for miR‐193a‐3p. K, Western blot of TGFβR3 (94 kD), TGFβ2 (52 kD) and DR6 (23.7 kD) proteins extracted from splenocytes transfected with mimic or inhibitor of miR‐193a or mock. Numbers below blots indicate the densitometry of the bands when normalized to GAPDH protein. Significance was depicted as follows: *P < .05, **P < .01, ***P < .001, ****P < .0001

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