Multiple anti-inflammatory pathways triggered by resveratrol lead to amelioration of staphylococcal enterotoxin B-induced lung injury

Abstract

Background and purpose: Inhalation of the superantigen,staphylococcal enterotoxin B (SEB), leads to the activation of the host T and invariant natural killer (iNK) T cells, thereby resulting in acute lung inflammation and respiratory failure but the underlying mechanism(s) of disease remain elusive, with limited treatment options. In this study, we investigated the therapeutic effectiveness of resveratrol, a plant polyphenol, during SEB-induced lung inflammation.

Experimental approach: C57BL/6 mice were exposed to SEB (50 µg·per mouse), administered intranasally, and were treated with resveratrol (100 mg·kg(-1)) before or after SEB exposure. Lung injury was studied by measuring vascular permeability, histopathological examination, nature of infiltrating cells, inflammatory cytokine induction in the bronchoalveolar fluid (BALF), apoptosis in SEB-activated T cells and regulation of SIRT1 and NF-κB signalling pathways.

Key results: Pretreatment and post-treatment with resveratrol significantly reduced SEB-induced pulmonary vascular permeability, and inflammation. Resveratrol significantly reduced lung infiltrating cells and attenuated the cytokine storm in SEB-exposed mice, which correlated with increased caspase-8-dependent apoptosis in SEB-activated T cells. Resveratrol treatment also markedly up-regulated Cd11b+ and Gr1+ myeloid-derived suppressor cells (MDSCs) that inhibited SEB-mediated T cell activation in vitro. In addition, resveratrol treatment was accompanied by up-regulation of SIRT1 and down-regulation of NF-κB in the inflammatory cells of the lungs.

Conclusions and implications: The current study demonstrates that resveratrol may constitute a novel therapeutic modality to prevent and treat SEB-induced lung inflammation inasmuch because it acts through several pathways to reduce pulmonary inflammation.

© 2012 The Authors. British Journal of Pharmacology © 2012 The British Pharmacological Society.

Figures

Figure 1

Resveratrol treatment alleviates SEB-induced capillary leak and inflammation in the lungs. (A) Timeline for the in vivo studies. SEB (50 µg·per mouse) was administered to mice by the intranasal route on day 0. Mice in the pretreatment group received a single oral dose of resveratrol (RES; 100 mg·kg−1 body weight in sterile water) one day before SEB exposure, as well as on days 0 and 1. Mice in the post-treatment group received a single oral dose of resveratrol on day 0 (30 min after SEB administration) and on day 1. Mice were killed on day 2 to study vascular leak and histopathology. (B) Measurement of capillary leak in the lungs using Evan's Blue dye extravasation. Percent increase in vascular leak was calculated compared to control. (C) Haematoxylin and eosin staining of lung sections from different treatment groups of mice. The photographs were taken at 40× and 100×. (D) Quantification of the histopathology data. Layers of infiltrating cells were counted around 10 different capillaries of the same size, and the data represent the average for each individual group. Vertical bars represent data collected from five mice per group expressed as mean ± SEM. anova, P < 0.0006 with Tukey's test; ***P < 0.05, significantly different from RES only, †P < 0.05, significantly different from SEB only.

Figure 2

Resveratrol treatment reduces inflammatory cell populations in SEB-exposed lungs. (A) Representative dot-plots for the phenotyping of cells in the lungs. Mice were treated as stated in Figure 1A, and on day 2, lungs were excised, and immune cells were isolated. Cells were then stained with various markers. (CD3+: T cells, NK1.1+: NK cells, CD3+NK1.1+: NKT cells, Gr1+: granulocytes, Cd11b+: primarily macrophages and to a lower extent in granulocytes, NK cells and a subset of DCs, Gr1+Cd11b+: MDSCs) (B) Total number of cells isolated from the lungs of mice. Numbers represent the total per mouse. Vertical bars represent data collected from five mice per group expressed as mean ± SEM. anova: P < 0.001 with Tukey's test; ***P < 0.05, significantly different from Control, ‡P < 0.05, significantly different from SEB only.

Figure 2

Resveratrol treatment reduces inflammatory cell populations in SEB-exposed lungs. (A) Representative dot-plots for the phenotyping of cells in the lungs. Mice were treated as stated in Figure 1A, and on day 2, lungs were excised, and immune cells were isolated. Cells were then stained with various markers. (CD3+: T cells, NK1.1+: NK cells, CD3+NK1.1+: NKT cells, Gr1+: granulocytes, Cd11b+: primarily macrophages and to a lower extent in granulocytes, NK cells and a subset of DCs, Gr1+Cd11b+: MDSCs) (B) Total number of cells isolated from the lungs of mice. Numbers represent the total per mouse. Vertical bars represent data collected from five mice per group expressed as mean ± SEM. anova: P < 0.001 with Tukey's test; ***P < 0.05, significantly different from Control, ‡P < 0.05, significantly different from SEB only.

Figure 3

Effect of resveratrol treatment on MDSCs in the lungs. (A) Number of MDSCs in the lungs. Mice were treated as stated in Figure 1A, and on day 2, lungs were excised, and immune cells were isolated. Cells were then stained with antibodies against Cd11b and Gr1 (Figure 2A). Absolute cell numbers were calculated per mouse and depicted as vertical bars (means ± SEM from five mice per group). *P < 0.005, ***P < 0.0001, significantly different from Control, Student's t-test. (B) MDSCs were further analysed for Ly6C surface expression. (C) Arginase I expression in the lung lymphocytes was determined by Western blot analysis 12 h after SEB administration. (D) Cell mixing experiment to study immunosuppressive properties of MDSCs. Axillary and inguinal lymph node cells were activated with 1 µg·mL−1 SEB and placed in culture with different populations of FACS Aria sorted MDSCs. Cell proliferation was measured at 48 h using thymidine incorporation assay. anova: P < 0.0001 with Tukey's test; ¥P < 0.05, significantly different from T cells only; *P < 0.05, **P < 0.01, ***P < 0.001, significantly different from T cells + SEB. (E) Wright–Giemsa stain of MDSCs that were sorted with FACS Aria.

Figure 4

Resveratrol treatment inhibits cytokine storm induced by SEB administration. Mice were treated as stated in Figure 1A and cytokine levels in (A) serum and (B) BAL fluid were measured on day 2, 48 h after SEB administration. Vertical bars represent data collected from five mice per group expressed as mean ± SEM. anova: P < 0.0001 with Tukey's test; ***P < 0.05, significantly different from Control, ‡P < 0.05, significantly different from SEB only.

Figure 5

Resveratrol (RES) induces apoptosis in SEB-activated lymphocytes via the extrinsic pathway of apoptosis. (A) Resveratrol induces cell death in a dose-dependent manner. Splenocytes from normal C57BL/6 mice were activated with SEB (1 µg·mL−1) either in the absence or presence of resveratrol (5, 10 and 20 µM). Twenty-four hours later, the cells were collected, washed and stained with TUNEL. In some experiments, inhibitors (20 µM) of caspase 8, 9 and 3 were added to the cultures 2 h before resveratrol treatment. (B) TUNEL studies with AhR (1 µM) and ER (1 µm) antagonists (α-naphthaflavone and tamoxifen respectively). Antagonists for AhR and ER were added 2 h before resveratrol treatment, and the cells were analysed by TUNEL assay 24 h after resveratrol treatment. This experiment was repeated at least three times, and the histograms show a representative experiment.

Figure 6

Resveratrol (RES) protects endothelial cells from cytotoxicity induced by SEB-activated lymphocytes. (A) Primary endothelial cells were isolated from the lungs of WT normal mice and expanded in culture for 10 days. Then, the endothelial cells were placed in culture with SEB-activated T cells for 24 h. T cells were then washed off, and the endothelial cells were stained with fluorometric TUNEL staining. (B) Quantification of the fluorimetric staining in panel A was performed by counting the apoptotic cells in 10 optical fields at 20× magnification. anova: P < 0.0001 with Tukey's test; ***P < 0.001, significantly different from endothelial cells only, ‡P < 0.05, significantly different from endothelial cells+ SEB T cells. (C) Primary endothelial cells were isolated from the lungs of normal mice and treated with TNF-α (5 ng·mL−1) and cycloheximide (0.5 µg·mL−1) overnight either in the absence or presence of resveratrol (5, 10 and 20 µM), and the cells were stained with anti-CD31, DAPI and TUNEL. White arrows indicate the cells that are undergoing apoptosis. (D) Quantification of the fluorimetric staining in panel C was performed by counting the apoptotic cells in 10 optical fields at 20× magnification. anova: P < 0.0001 with Tukey's test; ‡P < 0.05, significantly different from activated endothelial cells.

Figure 7

Resveratrol induces SIRT1 and inhibits NF-κB in the lungs (A) In vitro expression of SIRT1. Relative expression of SIRT1 was measured by real-time PCR. Naïve and SEB-activated splenocytes were placed in culture either in the absence or presence of resveratrol (RES; 5, 10 and 20 µM). Three hours later, the cells were collected, mRNA was isolated, cDNA was synthesized and RT-PCR was performed. (B) Western blot analysis of SIRT1. (C) The mice were treated as described in Figure 1A, and the lung lymphocytes were isolated 12 h after SEB administration. Next, mRNA was isolated, cDNA was synthesized and RT-PCR was performed. (D) Mice were treated as described in Figure 1A, and the lung lymphocytes were isolated with Ficoll-Hypaque gradients. The nuclear proteins were then separated, and used for NF-κB- p-65 Sandwich elisa assay. The results were calculated as % increase, relative to control. Correct fractionation was confirmed with blotting for lamin and γ-tubulin. anova: P < 0.01 with Tukey's test; ***P < 0.05, significantly different from Control, ‡P < 0.05, significantly different from SEB only.