Protective function of DJ-1/PARK7 in lipopolysaccharide and ventilator-induced acute lung injury

Abstract

Oxidative stress is considered one of the early underlying contributors of acute lung injury (ALI) and ventilator-induced lung injury (VILI). DJ-1, also known as PARK7, has a well-established role as an antioxidant. We have previously shown maintaining oxidative balance via the ATF3-Nrf2 axis was important in protection from ALI. Here, we exclusively characterize the role of DJ-1 in sterile LPS-induced ALI and VILI. DJ-1 protein expression was increased after LPS treatment in human epithelial and endothelial cell lines and lungs of wild-type mice. DJ-1 deficient mice exhibited greater susceptibility to LPS-induced acute lung injury as demonstrated by increased cellular infiltration, augmented levels of pulmonary cytokines, enhanced ROS levels and oxidized by-products, increased pulmonary edema and cell death. In a two-hit model of LPS and mechanical ventilation (MV), DJ-1 deficient mice displayed enhanced susceptibility to inflammation and lung injury. Collectively, these results identify DJ-1 as a negative regulator of ROS and inflammation, and suggest its expression protects from sterile lung injury driven by high oxidative stress.

Keywords: Acute lung injury; DJ-1; PARK7; VILI.

Graphical abstract

Fig. 1

Increased DJ-1 expression in human lung cells and mice following oxidative and inflammatory stimuli. Doses: LPS - μg/mL, TNF-α - ng/mL, H₂O₂ - μM. Representative western blots showing increased DJ-1 protein expression in A) A549 cells (n = 2 independent experiments), B) HPMEC cells (n = 2 independent experiments), C) Beas-2b cells with different stimuli (n = 3 independent experiments), and D) Beas-2b cells with different doses (n = 3 independent experiments), following stimulation normalized to β-ACTIN protein expression. E) Representative Western blot and showing increased DJ-1 protein expression in whole lung lysates, following LPS stimulation normalized to β-ACTIN protein expression. Densitometry measurements (protein/β-actin) are mean + SEM. *<0.05 vs. saline, #p < 0.05 vs. LPS 0.1, $p < 0.05 vs. TNF-α 1. F) Representative immunohistochemistry images of DJ-1 expression (DAB brown staining) in lungs following 24 h of LPS instillation.

Fig. 2

Absence of DJ-1 exacerbates LPS-induced acute lung injury. A) Percent survival of wildtype (WT) and DJ-1−/− mice at 7 days after LPS instillation compared with saline (SAL) controls. (saline n = 5, LPS n = 10–11, *p < 0.05 **p < 0.01, Log-rank/Mantel-Cox analysis) B) Representative H&E stained images of lungs 24 h post-instillation of SAL or LPS. C) Lung Injury Scores (LIS) (n = 3–5 animals) as characterized previously using alveolar septum thickening (AT), hemorrhagic infiltration (HI), exudative edema (EE), bronchus-associated lymphoid tissue aggregates (BALT), perivascular inflammatory infiltrates (PVI), and alveolar septal infiltration (ASI). D) Representative H & E stained images of Bronchoalveolar lavage fluid (BALF) cyto-spins from WT and DJ-1−/− mice exposed to saline or LPS treatment. BALF E) total cell count (n = 8–13), and F) percent neutrophils (n = 8–13) in WT and DJ-1−/− mice at 24 h after saline and LPS treatment. G) Myeloperoxidase levels in lung tissue lysates with saline or LPS treatment (n = 7–14). H) BALF total protein (n = 7–14) at 24 h after saline or LPS treatment. Data are presented as means ± SEM (*p < 0.05, **p < 0.01, ***p < 0.001, two-way ANOVA analysis).

Fig. 3

DJ-1 deficiency enhances lung inflammation following LPS instillation. Levels of inflammatory mediators in lung homogenates and serum from WT or DJ-1−/− mice 24 post LPS instillation. Mediators profiled: interleukin-1 beta (IL-1β), macrophage inflammatory protein 1 alpha (MIP-1α/CCL3), monocyte chemoattractant protein-1 (MCP-1/CCL2), interferon gamma (IFN-γ), keratinocyte chemoattractant (KC/CXCL1), LIX(CXCL5), interleukin-6 (IL-6) and interferon gamma-induced protein 10 (IP-10/CXCL10. Data are presented as means ± SEM (n = 6–8 per group, *p < 0.05, **p < 0.01, two-way ANOVA).

Fig. 4

Loss of DJ-1 aggravates pulmonary oxidative stress following LPS stimulation. A) Representative images of H₂DCFDA-loaded endothelial cells in micro-vessels of the isolated perfused mouse lung showing imaging of endothelial DCF with increasing fluorescence intensity in the DJ-1−/− B) and quantitative analysis of DCF fluorescence relative to baseline showing increased ROS production in response to TNF-α infusion (1000 U/mL) in DJ-1−/− compared to WT lungs. Data are presented as means ± SEM (n = 3–5 mice per group, *p < 0.05 compared to WT saline and #p < 0.05 compared to WT TNF treated). 8-Isoprostane levels in C) lung lysates and D) BALF in WT and DJ-1−/− animals following LPS instillation. Bar graphs represent means ± SEM (n = 7–9 per group, *p ≤ 0.05, **p ≤ 0.01, two-way ANOVA). Real-time PCR analysis of E) HMOX-1, F) NQO-1 and G) GPX-1 mRNA levels in WT and DJ-1−/− lungs 24 h following LPS instillation. Bar graphs represent means ± SEM (n = 6–8 per group, *p ≤ 0.05, **p ≤ 0.01, two-way ANOVA).

Fig. 5

Loss of DJ-1 leads to oxidative stress-induced enhanced cell death. A) Western blots showing protein expression of NRF2, HMOX1, NADPH OXIDASE 2 (NOX2), JNK, pJNK, caspase 3 (CASP3), cleaved caspase 3 (CL-CASP3), DJ-1 and B-ACTIN. Densitometry measurements (protein/β-actin) mean + SEM. n = 2–3. *p < 0.05 vs. WT saline, #p < 0.05 vs. WT LPS, $p < 0.05 vs. DJ-1−/− saline. Cell death analysis using Lactate dehydrogenase (LDH) release in B) BALF (n = 6–8 animals each) and C) supernatants of Beas-2b cells treated with 50 nM control siRNA or DJ-1 siRNA (n = 3 independent experiments. D) Propidium Iodide (PI) staining (n = 3 independent experiments) and E) Annexin V staining in control and DJ-1 siRNA treated Beas-2b cells (n = 1 independent experiment). *p < 0.05, **p < 0.01, ***p < 0.001, two-way ANOVA.

Fig. 6

DJ-1 deficiency exacerbated increased lung injury and impaired function in LPS + MV model. A) Percent survival of WT and DJ-1−/− mice at 4 h after saline or LPS instillation with or without mechanical ventilation (MV) (*p < 0.05 Log-rank/Mantel-Cox analysis). B) Representative images of whole lungs from WT and DJ-1−/− mice following LPS + mechanical ventilation (MV). C) Representative images of H & E stained BALF cytospins from WT and DJ-1−/− mice at 4 h after saline or LPS instillation with or without mechanical ventilation (MV). BALF D) total cell count, E) percent neutrophils and F) total protein in WT and DJ-1−/− mice at 4 h post LPS and ventilation. Data are presented as means ± SEM (n = 6–9 saline and 11–17 LPS mice) *p < 0.05 compared with saline control of respective genotype $p < 0.05 compared with non-ventilated LPS group, #p < 0.05 compared with WT LPS ventilated, two-way ANOVA analysis).

Fig. 7

DJ-1 deficient animals had increased expression of oxidative stress and cell death markers in a “two-hit” model. A) mRNA Expression of Heme oxygenase-1(HMOX-1), B) NADPH quinone oxidoreductase 1 (NQO1), C) inducible NOS (iNOS), D) and FAS in WT and DJ-1−/− mechanically ventilated (MV) mice with either saline or LPS administration. Data are presented as means ± SEM (n = 6–8 mice, *p < 0.05, two-way ANOVA). E) Western blots showing protein expression of Nrf2, NADPH oxidase 2 (NOX2), p47phox, FAS, p53, JNK, pJNK, cleaved caspase-3, DJ-1 and B-ACTIN. Densitometry measurements (protein/β-actin) are mean + SEM. n = 2–4. *p < 0.05 vs. WT saline (SAL) + MV, #p < 0.05 vs. WT LPS + MV, $p < 0.05 vs. DJ-1−/− SAL + MV. F) Western blot showing immunoprecipitation with Nrf2 and probing for Keap1 or Ubr7 in Beas-2b cells with control or DJ-1 siRNA (n = 2 independent experiments).