Nrf2 Lowers the Risk of Lung Injury via Modulating the Airway Innate Immune Response Induced by Diesel Exhaust in Mice

Abstract

In the present study, we investigated the role of Nrf2 in airway immune responses induced by diesel exhaust (DE) inhalation in mice. C57BL/6J Nrf2^+/+ and Nrf2^-/- mice were exposed to DE or clean air for 8 h/day and 6 days/week for 4 weeks. After DE exposure, the number of neutrophils and macrophage inflammatory protein (MIP)-2 level in bronchoalveolar lavage fluid (BALF) and interleukin (IL)-17 level in the lung tissue increased in Nrf2^-/- mice compared with Nrf2^+/+ mice; however, the lack of an increase in the level of tumor necrosis factor (TNF)-α in the lung tissue in Nrf2^+/+ mice and mild suppression of the level of TNF-α in Nrf2^-/- mice were observed; the level of granulocyte macrophage colony-stimulating factor (GM-CSF) in the lung tissue decreased in Nrf2^-/- mice than in Nrf2^+/+ mice; the number of DE particle-laden alveolar macrophages in BALF were larger in Nrf2^-/- mice than in Nrf2^+/+ mice. The results of electron microscope observations showed alveolar type II cell injury and degeneration of the lamellar body after DE exposure in Nrf2^-/- mice. Antioxidant enzyme NAD(P)H quinone dehydrogenase (NQO)1 mRNA expression level was higher in Nrf2^+/+ mice than in Nrf2^-/- mice after DE exposure. Our results suggested that Nrf2 reduces the risk of pulmonary disease via modulating the airway innate immune response caused by DE in mice.

Keywords: immune response; lung diseases; macrophage; neutrophils; oxidative stress/anti-oxidative stress.

Figure 1

Differential cell counts in bronchoalveolar lavage fluid (BALF). (A) total cells, (B) macrophages, (C) neutrophils, (D) lymphocytes. Data are shown as mean ± SD values in each group (Nrf2^+/+ control: n = 6; Nrf2^+/+ DE: n = 6; Nrf2^−/− control: n = 4; Nrf2^−/− DE: n =5), * p < 0.05 vs. control; # p < 0.05 vs. Nrf2^+/+.

Figure 2

The pathologic features of diesel exhaust (DE) particles (DEP)-laden alveolar macrophages in BALF. (A) DE group in Nrf2^+/+ mice; (B) DE group in Nrf2^−/− mice. Representative optical micrographs of May–Giemsa (Kobe, Japan) stained cell preparations. Scale bar was 200 μm. Arrows indicate DEP-laden alveolar macrophages. DEP-laden macrophages in which DEP accounted for more than half of the cytoplasm were defined as coal-black cells. Arrowhead indicates coal-black alveolar macrophages. (C) Percentages of DEP-laden macrophages and coal-black alveolar macrophages in total macrophages. Data are shown as mean ± SD values in each group (Nrf2^+/+ DE: n = 6; Nrf2^−/− DE: n = 5), # p < 0.05 vs. Nrf2^+/+.

Figure 3

Electron microscopic analysis of alveolar macrophages and alveolar type II epithelial cells (Type II cells). The photographs show representative results. Alveolar macrophages in Nrf2^+/+ mice, control group (A), DE group (B); alveolar macrophages in Nrf2^−/− mice, control group (C), DE group (D). Type II cells in Nrf2^+/+ mice, control group (E), DE group (F); Type II cells in Nrf2^−/− mice, control group (G), DE group (D,H). Black arrows indicate alveolar macrophages engulfing DEP (B), and degeneration of the lamellar body (F,H). A white dotted ring indicates the apoptotic body that was phagocytosed by a macrophage (B). M: macrophage; Type II: alveolar type II epithelial cells; *: irregular condensed chromatin and nuclear fragmentation; CSS: crescent-shaped spaces (appearing around the nucleus of an apoptotic cell); Apo: apoptosis; RBC: red blood cell; C: collagen fiber; L: lumen.

Figure 4

Macrophage inflammatory protein (MIP)-2 (A) and surfactant protein (SP)-D (B) in BALF were analyzed using ELISA. The vertical axis shows percent changes in the cytokines of the DE group relative to the control group. Data are shown as mean ± SD values in each group (n = 5). * p < 0.05 vs. control.

Figure 5

TNF-α (A), GM-CSF (B), IL-17 (C), and IL-33 (D) levels in lung tissue supernatants were analyzed using ELISA. Total protein concentrations in the supernatants were measured to normalize the concentration levels of target cytokines in the supernatants. The vertical axis shows percent changes in the cytokines of the DE group relative to the control group. Data are shown as mean ± SD values in each group (n = 5). * p < 0.05 vs. control; # p < 0.05 vs. Nrf2^+/+.

Figure 6

HO-1 (A) and NQO1 (B) mRNA expression levels in the lung tissues were analyzed using real time RT-PCR. The vertical axis shows percent changes in target gene mRNA expression levels of the DE group relative to the control group. β-actin was used as an internal control. Data are shown as mean ± SD values in each group (n = 3). * p < 0.05 vs. control; # p < 0.05 vs. Nrf2^−/−.

Figure 7

Schematic diagram of putative mechanism of oxidative stress and its effect on airway immune response to DE exposure in mice. DE: diesel exhaust; ROS: reactive oxygen species; NETs: neutrophil extracellular traps; GM-CSF: granulocyte macrophage colony-stimulating factor; Type II cells: alveolar type II epithelial cells; COPD: chronic obstructive pulmonary disease; ARDS: acute respiratory distress syndrome; Solid line: putative signaling pathways in the airway response to DEP exposure; Dashed line: unclear signaling pathways; Arrowhead: promoted in Nrf2^−/−; ↓: inhibited in Nrf2^−/−; ↑: stimulated in Nrf2^−/−.