The compromise of macrophage functions by hyperoxia is attenuated by ethacrynic acid via inhibition of NF-κB-mediated release of high-mobility group box-1

Abstract

The prolonged exposure to hyperoxia can compromise macrophage functions and contribute to the development of ventilator-associated pneumonia. High levels of extracellular high-mobility group box-1 (HMGB1) in the airways of mice exposed to hyperoxia can directly cause macrophage dysfunction. Hence, inhibition of the release of nuclear HMGB1 into the extracellular milieu may help to maintain macrophage functions under hyperoxic conditions. The present study investigates whether ethacrynic acid (EA) affects hyperoxia-induced HMGB1 release from macrophages and improves their functions. Macrophage-like RAW 264.7 cells and bone marrow-derived macrophages were exposed to different concentrations of EA for 24 hours in the presence of 95% O2. EA significantly decreased the accumulation of extracellular HMGB1 in cultured media. Importantly, the phagocytic activity and migration capability of macrophages were significantly enhanced in EA-treated cells. Interestingly, hyperoxia-induced NF-κB activation was also inhibited in these cells. To determine whether NF-κB plays a role in hyperoxia-induced HMGB1 release, BAY 11-7082, an inhibitor of NF-κB activation, was used. Similar to EA, BAY 11-7082 significantly inhibited the accumulation of extracellular HMGB1 and improved hyperoxia-compromised macrophage migration and phagocytic activity. Furthermore, 24-hour hyperoxic exposure of macrophages caused hyperacetylation of HMGB1 and its subsequent cytoplasmic translocation and release, which were inhibited by EA and BAY 11-7082. Together, these results suggest that EA enhances hyperoxia-compromised macrophage functions by inhibiting HMGB1 hyperacetylation and its release from macrophages, possibly through attenuation of the NF-κB activation. Therefore, the activation of NF-κB could be one of the underlying mechanisms that mediate hyperoxia-compromised macrophage functions.

Keywords: NF-κB; high-mobility group box-1; hyperoxia; macrophage; phagocytosis.

Figure 1.

Effect of ethacrynic acid (EA) on hyperoxia-induced high-mobility group box-1 (HMGB1) release from RAW 264.7 cells and bone marrow–derived macrophages (BMDMs). RAW 264.7 cells and BMDMs were exposed to 95% O₂ in the presence of different concentrations of EA for 24 hours. The levels of extracellular HMGB1 in the culture media of (A) RAW 264.7 cells and (B) BMDMs were determined by Western blot analysis. The levels of HMGB1 were quantified by measuring the integrated density value (IDV) of the immunoreactive bands on Western blots and expressed as % HMGB1, considering 95% O₂ as 100%. Each value represents the mean ± SEM of three and two independent experiments for each group in RAW 264.7 cells and BMDMs, respectively. *P < 0.05; **P < 0.01 compared with cells exposed to 95% O₂ in DMSO alone.

Figure 2.

Effects of EA on HMGB1 localization in hyperoxic RAW 264.7 cells. RAW 264.7 cells, grown on coverslips, were exposed to room air (21% O₂) or 95% O₂ in the presence of different concentrations of EA for 24 hours. HMGB1 localization was visualized by immunofluorescence microscopy (A), and quantification of fluorescence intensity (B) was performed using ImageJ software (National Institutes of Health, Bethesda, MD). Counterstaining with 4′,6-diamidino-2-phenylindole (DAPI) was used to visualize nuclei (insets). Each value represents the mean ± SEM of three independent experiments for each group. *P < 0.05, compared with cells exposed to 95% O₂ in DMSO alone.

Figure 3.

EA restores the phagocytic ability of macrophages exposed to hyperoxia. RAW 264.7 cells were exposed to room air (21% O₂) or 95% O₂ in the presence of different concentrations of EA for 24 hours. Cells were subsequently incubated with FITC-labeled latex minibeads for 1 hour and stained with phalloidin to visualize the cells. (A) shows representative immunofluorescence images of RAW 264.7 cells phagocytosing minibeads [bright dots]. At least 250 individual macrophages per well were counted to quantify the extent of phagocytic activity (B). Each value represents the mean ± SEM of three independent experiments for each group. *P < 0.05, compared with cells exposed to 95% O₂ in DMSO alone.

Figure 4.

Effects of EA on hyperoxia-suppressed macrophage migration. (A) RAW 264.7 cells were seeded and incubated overnight in 24-well plates. Cells were scratched and photographed immediately (0 h) as the 0 hour. Cells were then allowed to migrate in room air (21% O₂) or hyperoxia (95% O₂) in the presence of different concentrations of EA for 24 hours. The scratched regions were then photographed again at 24 hours to visualize the migration of cells (after treatment). (B) The graph shows the percentage of cells migrating to the wound regions. Each value represents the mean ± SEM of three independent experiments. *P < 0.05 compared with cells exposed to 95% O₂ in vehicle DMSO alone; ^#P < 0.05 compared with cells remained in room air.

Figure 5.

EA inhibits nuclear translocation of NF-κB p65 subunit and phosphorylation of p65 (p-p65) on serine 536 in hyperoxic RAW 264.7 cells. (A) RAW 264.7 cells, grown on coverslips, were exposed to room air (21% O₂) or to 95% O₂ in the presence of different concentrations of EA for 24 hours and analyzed for the localization of NF-κB p65 subunit by an immunofluorescence assay. After oxygen exposure, cells were washed with PBS, fixed, permeabilized, and stained to localize the NF-κB p65 subunit. The nuclear translocation of p65 was visualized using immunofluorescence microscopy. Counterstaining with DAPI was used to visualize nuclei (insets). (B) RAW 264.7 cells were exposed to room air (21% O₂) or to 95% O₂ in the presence of different concentrations of EA for 2 hours, and phosphorylation of p65 on serine 536 was assessed by Western blot analysis. Representative image of immunoreactive bands on Western blot is shown. Bar graph shows the integrated density value of p-p65 bands. The data are expressed as mean ± SEM of two independent experiments. *P < 0.05, compared with cells exposed to 95% O₂ in DMSO alone.

Figure 6.

BAY 11-7082 inhibits the hyperoxia-induced activation of NF-κB and HMGB1 release, and restores hyperoxia-suppressed macrophage functions. RAW 264.7 cells, grown on coverslips, were exposed to hyperoxia in the presence of different concentrations of BAY 11-7082 for 24 hours. (A and B) After exposure, cells were washed with PBS, fixed, permeabilized, and stained to localize (A) NF-κB p65 subunit or (B) HMGB1. The localization of the NF-κB p65 subunit or HMGB1 was visualized using immunofluorescence microscopy. Representative images are shown. (C) Levels of HMGB1 in the cell supernatants were determined by Western blot analysis. The levels of HMGB1 were quantified by measuring an integrated density value (IDV) in arbitrary units (AU) of the immunoreactive bands on Western blots. Each value represents the mean ± SEM of three independent experiments for each group. *P < 0.05, **P < 0.01 compared with cells exposed to 95% O₂ in DMSO alone. (D) RAW 264.7 cells, grown on coverslips, were exposed to room air (21% O₂) or 95% O₂ alone in the presence of different concentrations of BAY 11-7082 for 24 hours. Cells were subsequently incubated with FITC-labeled latex minibeads for 1 hour and stained with phalloidin and DAPI. At least 50 cells/slide were counted to quantify the extent of phagocytosis. Each value represents the mean ± SEM of three independent experiments for each group. *P < 0.05, **P < 0.01 compared with cells exposed to 95% O₂ in DMSO alone. (E) RAW 264.7 cells were seeded and incubated overnight in 24-well plates. Cells were scratched and photographed immediately (0 h). The cells remained in room air (21% O₂) or were exposed to 95% O₂ in the presence of different concentrations of BAY 11-7082 for 24 hours. The scratched regions were photographed again to visualize migration of cells (after treatment). The graph shows the percentage of migrating cells to the wound region. Each value represents the mean ± SEM of three independent experiments. *P < 0.05, **P < 0.01 compared with cells exposed to 95% O₂ in DMSO alone.

Figure 7.

EA and BAY 11-7082 inhibit hyperoxia-induced acetylation of HMGB1. Representative spectra of the liquid chromatography mass spectrometric (LC-MS) characterization of peptides produced from HMGB1 derived from macrophage cell lysates (A) and supernatants (B) enzymatically cleaved with endopeptidase Staphylococcus aureus protease V8 (Gluc). Macrophages were exposed to 21 or 95% O₂ in the presence or absence of either EA or BAY 11-7082. The presence of the peptides with molecular weights 1,624 and 1,133 Da indicate the hypoacetylation of lysine residues within nuclear localization signal (NLS) 1 and 2, respectively (n = 1). The presence of the peptides with molecular weights 1,750 and 1,343 Da indicate the hyperacetylation of lysine residues within NLS 1 and 2, respectively. The experiment was performed once. (C) Proposed schematic overview of the mechanism of hyperoxia-induced HMGB1 acetylation and its inhibition by EA or BAY 11-7082.

Figure 8.

A hypothesized pathway indicating how NF-κB may modulate macrophage functions via its effect on hyperoxia-induced HMGB1 release. Under normoxic conditions, macrophages can efficiently phagocytose invading pathogens. HMGB1 regularly shuttles between the nucleus and the cytoplasm, depending on its acetylation status. Whereas underacetylated HMGB1 is imported into the nucleus, hyperacetylation of HMGB1 promotes its export to the cytoplasm. Under normoxic conditions, HMGB1 is underacetylated. Thus, the nuclear import predominates the export, resulting in its prevalent localization in the nucleus. The prolonged exposure of macrophages to hyperoxia inhibits their phagocytic function and causes HMGB1 release into the extracellular milieu. Before its release, HMGB1 translocates from the nucleus to the cytoplasm as a result of its hyperacetylation upon prolonged exposure to hyperoxia. The exposure of macrophages to hyperoxia also leads to the translocation of NF-κB into the nucleus, indicative of its activation. Nuclear NF-κB has been shown to activate histone acetyltransferases (HATs) (72), which can hyperacetylate HMGB1, thereby favoring its translocation to the cytoplasm and, subsequently, its extracellular release. EA and BAY 11-7082 significantly inhibited the activation of NF-κB, which further inhibited hyperoxia-induced release of acetylated HMGB1 and its translocation. It is likely that NF-κB modulates macrophage functions by hyperacetylation of HMGB1, thereby inhibiting its release. Solid arrows represent known pathways; dashed arrows represent possible pathways.