Genetic ablation of NADPH oxidase enhances susceptibility to cigarette smoke-induced lung inflammation and emphysema in mice

Abstract

Cigarette smoke (CS) induces recruitment of inflammatory cells in the lungs leading to the generation of reactive oxygen species (ROS), which are involved in lung inflammation and injury. Nicotinamide adenine dinucleotide phosphate (NADPH) oxidase is a multimeric system that is responsible for ROS production in mammalian cells. We hypothesized that NADPH oxidase-derived ROS play an important role in lung inflammation and injury and that targeted ablation of components of NADPH oxidase (p47(phox) and gp91(phox)) would protect lungs against the detrimental effects of CS. To test this hypothesis, we exposed p47(phox-/-) and gp91(phox-/-) mice to CS and examined inflammatory response and injury in the lung. Surprisingly, although CS-induced ROS production was decreased in the lungs of p47(phox-/-) and gp91(phox-/-) mice compared with wild-type mice, the inflammatory response was significantly increased and was accompanied by development of distal airspace enlargement and alveolar destruction. This pathological abnormality was associated with enhanced activation of the TLR4-nuclear factor-kappaB pathway in response to CS exposure in p47(phox-/-) and gp91(phox-/-) mice. This phenomenon was confirmed by in vitro studies in which treatment of peritoneal macrophages with a nuclear factor-kappaB inhibitor reversed the CS-induced release of proinflammatory mediators. Thus, these data suggest that genetic ablation of components of NADPH oxidase enhances susceptibility to the proinflammatory effects of CS leading to airspace enlargement and alveolar damage.

Figure 1

Increased neutrophil and decreased macrophage counts in the BAL fluid of p47^phox−/− and gp91^phox−/− mice in response to CS exposure. The number of neutrophils in BAL fluid from 3-day (A) and 8-week (B) air- or CS-exposed mice. The number of neutrophils in BAL fluid was significantly increased in p47^phox−/− and gp91^phox−/− mice compared to WT mice in response to CS exposure. Lavaged macrophages from 3-day (C) and 8-week (D) air- or CS-exposed mice. The number of macrophages in BAL fluid was significantly decreased in gp91^phox−/− mice compared to WT mice in response to CS exposure. Data are shown as mean ± SEM (n = 3 to 4 per group). *P < 0.05, **P < 0.01, ***P < 0.001, significant compared with respective air-exposed groups; ⁺P < 0.05, ⁺⁺P < 0.01, ⁺⁺⁺P < 0.001, significant compared with CS-exposed WT mice; ^#P < 0.05, significant compared with air-exposed WT mice.

Figure 2

Increased inflammatory cell influx into the lungs of p47^phox−/− and gp91^phox−/− mice exposed to CS. Figure shown is H&E-stained lung sections from 3-day (A) and 8-week (B) air- or CS-exposed mice (n = 3 per group). Lung sections from p47^phox−/− and gp91^phox−/− mice show increased inflammatory cell infiltration (A and B) and alveolar destruction and airspace enlargement (B) compared to WT mice. Black arrows indicate inflammatory cells, white arrows indicate alveolar destruction. *P < 0.05, **P < 0.01, significant compared with respective air-exposed groups; ⁺P < 0.05, ⁺⁺P < 0.01, significant compared with CS-exposed WT mice; ^#P < 0.05, ^##P < 0.05, significant compared with air-exposed WT mice. Original magnifications, ×200.

Figure 3

Increased macrophage influx into lungs of p47^phox−/− and gp91^phox−/− mice exposed to CS. Immunohistochemical detection of macrophages (arrows) using rat anti-mouse Mac-3 antibody in lung from 3-day (A) and 8-week (B) air- or CS-exposed mice. The lung sections from CS-exposed p47^phox−/− and gp91^phox−/− mice showed more macrophages than those from CS-exposed WT counterparts. In addition, there were more alveolar macrophages in air-exposed p47^phox−/− and/or gp91^phox−/− mice than those from air-exposed WT mice. The pictures represent at least three separate experiments. **P < 0.01, ***P < 0.001, significant compared with respective air-exposed groups; ⁺P < 0.05, ⁺⁺P < 0.01, significant compared with CS-exposed WT mice; ^#P < 0.05, ^##P < 0.01, significant compared with air-exposed WT mice. Original magnifications, ×200.

Figure 4

Genetic ablation of p47^phox and gp91^phox increased lung activity of MPO in response to CS exposure. The activity of MPO in lungs was significantly increased in p47^phox−/− and gp91^phox−/− mice as compared to WT mice in response to 3-day (A) and 8-week (B) CS exposure. Data are shown as mean ± SEM (n = 3 to 4 per group). *P < 0.05, ***P < 0.001, significant compared with respective air-exposed group; ⁺P < 0.05, ⁺⁺⁺P < 0.001, significant compared with CS-exposed WT mice; ^#P < 0.05, significant compared with air-exposed WT mice.

Figure 5

Increased lung proinflammatory mediators release in p47^phox−/− and gp91^phox−/− mice exposed to CS. A: Acute CS exposure increased the levels of KC, MCP-1, IL-6, and TNF-α in the lungs of WT mice that were significantly increased in p47^phox−/− and gp91^phox−/− mice. B: Lung levels of GM-CSF, MCP-1, and KC were significantly increased in p47^phox−/− and gp91^phox−/− mice compared to WT mice exposed to CS for 8 weeks. A and B: Genetic ablation of p47^phox and gp91^phox also led to increased basal levels of GM-CSF, MCP-1, KC, and IL-6 in the lungs. The levels of proinflammatory mediators were measured by the Luminex¹⁰⁰ using the Beadlyte mouse multicytokine beadmaster kit. Data are shown as mean ± SEM (n = 4 to 5 per group). *P < 0.05, **P < 0.01, ***P < 0.001, significant compared with respective air-exposed group; ⁺⁺P < 0.01, ⁺⁺⁺P < 0.001, significant compared with CS-exposed WT mice; ^#P < 0.05, ^##P < 0.01, ^###P < 0.001, compared with air-exposed WT mice.

Figure 6

Increased mucus production shown by PAS staining in the airways of p47^phox−/− and gp91^phox−/− mice compared to WT mice in response to 8 weeks of CS exposure. Targeted disruption of p47^phox and gp91^phox increased mucus production in response to 8 weeks of CS exposure. In addition, increased mucus production was seen in the airways of air-exposed p47^phox−/− and gp91^phox−/− mice compared to air-exposed WT mice. The PAS staining pictures shown are from a representative experiment with three to four mice per group. Arrows indicate the mucus (purple) in the airway. *P < 0.05, significant compared with respective air-exposed group; ⁺P < 0.05, significant compared with CS-exposed WT mice; ^#P < 0.05, compared with air-exposed WT mice. Original magnifications, ×200.

Figure 7

ROS production is reduced in BAL cells of p47^phox−/− and gp91^phox−/− mice in response to CS exposure. Genetic ablation of p47^phox and gp91^phox decreased ROS production in BAL cells in response to 3-day (A) and 8-week (B) CS exposure. ROS production (arrows) in BAL cells was detected with DCF-DA fluorescence. The pictures shown are from a representative experiment with three mice per group. *P < 0.05, **P < 0.01, ***P < 0.001, significant compared with respective air-exposed group; ⁺P < 0.05, ⁺⁺P < 0.01, ⁺⁺⁺P < 0.001, significant compared with CS-exposed WT mice. Original magnifications, ×400.

Figure 8

Levels of lipid peroxidation products (4-HNE and MDA) are decreased in lungs of p47^phox−/− and gp91^phox−/− mice exposed to CS. The levels of 4-HNE and MDA were significantly reduced in p47^phox−/− and gp91^phox−/− mice compared to WT mice exposed to 3 days (A) and 8 weeks (B) of CS. Data are shown as mean ± SEM (n = 4 to 5 per group). **P < 0.01, ***P < 0.001, significant compared with respective air-exposed mice; ⁺P < 0.05, ⁺⁺⁺P < 0.001, significant compared with CS-exposed WT mice.

Figure 9

8-OHdG expression is decreased in lungs of p47^phox−/− and gp91^phox−/− mice exposed to CS. Immunohistochemical detection 8-OHdG (arrows) using anti-mouse monoclonal antibody in lungs of mice exposed to air or CS for 8 weeks. The lung sections from CS-exposed p47^phox−/− and gp91^phox−/− mice showed decreased 8-OHdG-positive cells than those from CS-exposed WT counterparts. The pictures are representative of at least three separate experiments. **P < 0.01, ***P < 0.001, significant compared with respective air-exposed groups; ⁺⁺P < 0.01, significant compared with CS-exposed WT mice. Original magnifications, ×200.

Figure 10

Increased levels of RelA/p65 and its phosphorylation on ser276 and ser536 in lung nuclear protein of p47^phox−/− and gp91^phox−/− mice in response to 3 days and 8 weeks of CS exposure. Acute and subchronic CS exposure increased the levels of RelA/p65 and its phosphorylation on ser276 and ser536 in lung nuclear protein of WT mice that were further increased in p47^phox−/− and gp91^phox−/− mice. In addition, the basal levels of RelA/p65 and its phosphorylation at ser276 and ser536 in the lungs were significantly increased in p47^phox−/− and gp91^phox−/− mice as compared to air-exposed WT mice. Gel pictures shown are representative of at least three separate experiments. The protein levels of RelA/p65 and its phosphorylation on ser276 and ser536 were assayed by Western blotting.

Figure 11

Degradation of IκBα in lung cytoplasmic fraction is increased in p47^phox−/− and gp91^phox−/− mice as compared to WT mice in response to 3 days and 8 weeks of CS exposure. Acute and subchronic CS exposure led to degradation of IκBα in lung cytoplasmic fraction that was increased in p47^phox−/− and gp91^phox−/− mice. Moreover, the basal level of IκBα in the lungs was significantly decreased in p47^phox−/− and gp91^phox−/− mice as compared to air-exposed WT mice. Gel pictures shown are representative of at least three separate experiments. Protein levels of IκBα were determined by Western blotting.

Figure 12

Increased levels of MCP-1 and KC in peritoneal macrophages from p47^phox−/− and gp91^phox−/− in response to CSE treatment that was partially inhibited by NF-κB inhibitor. Peritoneal macrophages were isolated from WT, p47^phox−/−, and gp91^phox−/− mice, and treated with CSE (0.25%, 0.5%, and 1%) in the presence or absence of NF-κB inhibitor (BAY-117082). CSE (0.25%, 0.5%, and 1%) induced the release of MCP-1 and KC from peritoneal macrophages of WT that was further increased in peritoneal macrophages derived from p47^phox−/− and gp91^phox−/− mice. Treatment of peritoneal macrophages with the NF-κB inhibitor significantly decreased CSE-mediated release of MCP-1 and KC. Data are shown as mean ± SEM (n = 4 to 5 per group). **P < 0.01, ***P < 0.001, significant compared with respective control group; ⁺P < 0.05, ⁺⁺P < 0.01, ⁺⁺⁺P < 0.001, significant compared with respective medium or CSE-treated peritoneal macrophages from WT mice; ^#P < 0.05, ^##P < 0.01, compared with respective CSE (0.5%)-treated group.

Figure 13

Increased TLR4 mRNA and protein expression in lung of p47^phox−/− and gp91^phox−/− mice in response to CS exposure. Genetic knockout of gp91^phox and p47^phox increased lung levels of TLR4 mRNA (A) and protein (B) as compared to WT mice in response to CS exposure. The levels of TLR4 mRNA (A) and protein (B) were also increased in air-exposed p47^phox−/− and/or gp91^phox−/− mice compared to air-exposed WT mice. Results shown are from a representative with three to four mice per group. The TLR4 mRNA and protein was assayed by real-time RT-PCR and Western blotting, respectively. *P < 0.05, **P < 0.01, ***P < 0.001, significant compared with respective air-exposed group; ⁺P < 0.05, ⁺⁺P < 0.01, ⁺⁺⁺P < 0.001, significant compared with CS-exposed WT mice; ^#P < 0.05, ^##P < 0.01, compared with air-exposed WT mice.

Figure 14

CSE causes the interaction of TLR4 with p47^phox and gp91^phox in peritoneal macrophages obtained from WT mice but this interaction is abrogated in p47^phox−/− and gp91^phox−/− mice. Peritoneal macrophages were isolated from WT, p47^phox−/−, and gp91^phox−/− mice, and treated with CSE (0.5%). CSE caused the interaction of TLR4 with p47^phox (A) and gp91^phox (B) in peritoneal macrophages from WT mice but this interaction was abrogated in p47^phox−/− and gp91^phox−/− mice, respectively. Gel pictures shown represent at least three separate experiments. The interaction of TLR4 with p47^phox and gp91^phox in peritoneal macrophages was analyzed using immunoprecipitation assay.

Figure 15

Increased levels of TLR4 protein in peritoneal macrophages from p47^phox−/− and gp91^phox−/− mice in response to CSE treatment. The levels of TLR4 protein were significantly increased in peritoneal macrophages obtained from p47^phox−/− and gp91^phox−/− compared to that from WT mice in response to CSE (0.25%, 0.5%, and 1%) treatments. Moreover, genetic ablation of p47^phox and gp91^phox increased the basal level of TLR4 protein. Results shown are from a representative of three separate experiments. The level of TLR4 protein was assayed with Western blotting. *P < 0.05, ***P < 0.001, significant compared with respective control group; ⁺⁺P < 0.01, ⁺⁺⁺P < 0.001, significant compared with respective CSE-treated peritoneal macrophages from WT mice; ^#P < 0.05, compared with control group in WT mice.

Figure 16

Compensatory regulation of NADPH oxidase subunits in lung of p47^phox−/− and gp91^phox−/− mice in response to CS exposure. Genetic knockout of gp91^phox and p47^phox increased the lung levels of Nox4 mRNA (A) and protein (B) at 3 days and 8 weeks by CS exposure as compared to WT mice. However, the mRNA (A) and protein (B) levels of p47^phox, gp91^phox, and/or Nox3 were not altered in p47^phox−/− and/or gp91^phox−/− mice exposed to CS as compared to WT mice. CS exposure led to an increase in mRNA and protein of p47^phox, gp91^phox, Nox3, and Nox4 in lungs of WT mice. Results shown are from a representative with three to four mice per group. The TLR4 mRNA and protein was assayed by RT-PCR and Western blotting, respectively.