GAT107-mediated α7 nicotinic acetylcholine receptor signaling attenuates inflammatory lung injury and mortality in a mouse model of ventilator-associated pneumonia by alleviating macrophage mitochondrial oxidative stress via reducing MnSOD-S-glutathionylation

Figures

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  Fig. 1. Systemic administration of GAT107 increases probability of survival and attenuates inflammatory lung injury in mice exposed to hyperoxia and PA lung infection. Male C57BL/6 were exposed to either 21% O2 (room air) or >95% O2 (hyperoxia) for 72 h, inoculated with PA (0.1 × 108 CFUs/mouse), and then returned to an environment of 21% O2 after inoculation. Hyperoxic mice were randomized to receive either GAT107 (3.3 mg/kg) or vehicle control intraperitoneally, every 12 h, starting at hour 24 of hyperoxia. (A) After mice were inoculated, animal survival was monitored and recorded every hour for 24 h and the percentage of mice that survived is shown in the Kaplan-Meier curve. Data was analyzed using the log-rank Mantel-Cox test (n = 5–10 mice/group). (B) BAL samples were harvested 24 h after PA inoculation. The total protein content in the BAL samples was quantified using bicinchoninic acid (BCA) assay. (C) The number of leukocytes was determined in BAL samples using a hemocytometer. (D) A differential cell counting analysis was used to determine the number of neutrophils following the staining of neutrophils with cytospin and Hema3 staining. Data represents the mean ± SEM from…

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  Fig. 2. GAT107 significantly attenuates hyperoxia-induced accumulation of extracellular HMGB1 and HMGB1-induced macrophage dysfunction. (A) Male C57BL/6 mice were exposed to either 21% O2 (room air) or >95% O2 (hyperoxia) for 48 h and then inoculated with PA (0.1 × 108 CFUs/mouse) and were returned to 21% O2 after inoculation. Hyperoxic mice were randomized to receive GAT107 (3.3 mg/kg) or vehicle control (1 mL/kg), intraperitoneally, every 12 h starting at hour 24 of hyperoxia. BAL was harvested 24 h after inoculation and western blot assays were conducted to determine the levels of extracellular HMGB1 in BAL. A representative western blot of HMGB1 is presented and HMGB1 levels were quantified using western blot analysis. (B) RAW 264.7 macrophages were either cultured in room air (white bar) or exposed to hyperoxia (black bar) without or with GAT107 in DMSO (grey bars). After 24 h, cell culture supernatant was collected and the level of extracellular HMGB1 was determined using western blot analysis. A representative western blot of HMGB1 is presented (B) and HMGB1 levels were quantified using western blot analysis. (C–D) RAW 264.7 macrophages were either exposed to vehicle control…

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  Fig. 3. GAT107 significantly attenuates hyperoxia-induced NF-kB activation and increases extracellular TNFa levels. RAW 264.7 macrophages were either exposed to 21% O2 (room air, white bar) or >95% O2 (hyperoxia, black bar) without or with GAT107 (grey bars). After 24 h, (A) cell lysates were collected and analyzed for NF-kB (p65 subunit) binding activity to DNA by ELISA using a NF-kB Transcription Factor Activity Kit and the results are represented as a bar graph; (B) cell culture supernatant was collected and analyzed for the levels of TNFa using ELISA. The bar graph represents the relative amounts of TNFa present in the cell culture media, compared to vehicle control. Each value represents the mean ± SEM of three independent experiments for each group. *p < 0.05, versus 0 μM (vehicle control).

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  Fig. 4. GAT107 decreases hyperoxia-induced mitochondrial membrane hyperpolarization and the accumulation of mitochondrial superoxide. RAW 264.7 macrophages were either exposed to 21% O2 (room air, white bar) or 95% O2 (hyperoxia, black bar) without or with GAT107 (grey bars). After 24 h, (A) macrophage mitochondrial integrity was determined using the tetramethylrhodamine, ethyl ester (TMRE) mitochondrial membrane potential assay that was normalized to MitoTracker Green. (B) The levels of mitochondrial superoxide in macrophages were analyzed using the MitoSOX dye assay at 24 h of hyperoxia. Each value represents mean ± SEM of three independent experiments for each group. *p < 0.05, versus the hyperoxia exposed vehicle treated control group.

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  Fig. 5. GAT107 attenuates hyperoxia-compromised MnSOD activity. RAW 264.7 macrophages were either exposed to 21% O2 (room air, white bar) or 95% O2 (hyperoxia, black bar) without or with GAT107 (grey bars). After 24 h, whole cell lysates were analyzed for total MnSOD levels using western blot assays for MnSOD. (A) Representative western blots for MnSOD and actin are presented, and (B) MnSOD and actin levels were quantified using western blot analysis. (C) RAW 264.7 macrophage cell lysate was used to determine MnSOD activity, using a SOD activity kit in the presence of cyanide. Each value represents mean ± SEM of three independent experiments for each group. #p < 0.05, versus the room air control group; *p < 0.05, versus the hyperoxia exposed vehicle treated control group.

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  Fig. 6. The hyperoxia-induced glutathionylation of MnSOD was significantly decreased by GAT107. RAW 264.7 macrophages were either exposed to 21% O2 (room air, white bar) or 95% O2 (hyperoxia, black bar) without or with GAT107 (grey bars). After 24 h, whole cell lysates were co-immunoprecipitated for MnSOD. (A) Representative western blots for immunoprecipitated MnSOD and conjugated GSH. (B) Densiometric analysis for immunoreactive GSH and MnSOD bands were determined and the ratio of glutathione (GSH) to MnSOD was reported as a percentage on the bar graph. Each value represents the mean ± SEM of 3–4 independent experiments for each group. *p < 0.05, versus hyperoxia exposed vehicle control group. (C) MnSOD activity of the samples compared to their corresponding percentage of MnSOD glutathionylation presented as a correlation plot. Each value on the correlation plot represents individual sample data points from 3 to 4 independent experiments. The measure of strength between the X and Y variables was determined by a two-tailed Pearson-correlation coefficient, which computed a p < 0.0001 between MnSOD activtabity and percentage of MnSOD glutathionylation.