Impairment of Mitochondrial ATP Synthesis Induces RIPK3-dependent Necroptosis in Lung Epithelial Cells During Lung Injury by Lung Inflammation

Abstract

Dysfunction of mitochondrial metabolism is implicated in cellular injury and cell death. While mitochondrial dysfunction is associated with lung injury by lung inflammation, the mechanism by which the impairment of mitochondrial ATP synthesis regulates necroptosis during acute lung injury (ALI) by lung inflammation is unclear. Here, we showed that the impairment of mitochondrial ATP synthesis induces receptor interacting serine/threonine kinase 3 (RIPK3)-dependent necroptosis during lung injury by lung inflammation. We found that the impairment of mitochondrial ATP synthesis by oligomycin, an inhibitor of ATP synthase, resulted in increased lung injury and RIPK3 levels in lung tissues during lung inflammation by LPS in mice. The elevated RIPK3 and RIPK3 phosphorylation levels by oligomycin resulted in high mixed lineage kinase domain-like (MLKL) phosphorylation, the terminal molecule in necroptotic cell death pathway, in lung epithelial cells during lung inflammation. Moreover, the levels of protein in bronchoalveolar lavage fluid (BALF) were increased by the activation of necroptosis via oligomycin during lung inflammation. Furthermore, the levels of ATP5A, a catalytic subunit of the mitochondrial ATP synthase complex for ATP synthesis, were reduced in lung epithelial cells of lung tissues from patients with acute respiratory distress syndrome (ARDS), the most severe form of ALI. The levels of RIPK3, RIPK3 phosphorylation and MLKL phosphorylation were elevated in lung epithelial cells in patients with ARDS. Our results suggest that the impairment of mitochondrial ATP synthesis induces RIPK3-dependent necroptosis in lung epithelial cells during lung injury by lung inflammation.

Keywords: Acute lung injury; Lung inflammation; Mitochondrial dysfunction; Necroptosis.

Figure 1. Impairment of ATP synthesis exacerbates lung injury by lung inflammation.

(A) Representative images for H&E staining and (B) quantification of lung injury scores of lung tissues from mice treated with oligomycin either control or LPS. Scale bars, 200 µm. Lung injury score was calculated by 100 fields from each group (n=20 images per individual subject, n=5 per each group). (C) Quantification of TNF-α levels in BALF from mice treated with oligomycin either control or LPS (n=5 per each group). Data are representative of three independent experiments. Data are mean±SD. ^**p<0.01; ^***p<0.001 by Student’s 2-tailed t-test, ANOVA or Kruskal-Wallis test.

Figure 2. Impairment of ATP synthesis increases the levels of RIPK3 in lung tissues during lung injury by lung inflammation.

(A) Representative immunoblot images of RIPK3 phosphorylation (Thr231/Ser232), RIPK3, MLKL phosphorylation (Ser345), MLKL, RIPK1 phosphorylation (Ser166) protein and (B) quantification of RIPK3 phosphorylation, RIPK3, MLKL phosphorylation, RIPK1 phosphorylation protein levels in lung tissues from mice treated with oligomycin either control or LPS (n=3 per each group). β-actin or MLKL were used as loading control. Data are representative of three independent experiments. Data are mean±SD. ^*p<0.05; ^**p<0.01; ^***p<0.001 by Student’s 2-tailed t-test or ANOVA.

Figure 3. Impairment of ATP synthesis induces the phosphorylation of MLKL in the activation of necroptosis during lung injury by lung inflammation.

(A) Representative immunofluorescence images for MLKL phosphorylation at Ser345 (P-MLKL) staining (green) in lung epithelial cells expressing E-cadherin (red) of mice treated with oligomycin either control or LPS. Scale bars, 20 µm. DAPI-stained nuclei are shown in blue. White arrows indicate P-MLKL positive cells (n=10 images per individual subject, n=5 per each group). (B) Quantification of intensity for P-MLKL-positive staining and (C) quantification of P-MLKL and E-cadherin-positive cells from immunofluorescence images in A. (D) Representative immunoblot images of RIPK3 phosphorylation (Thr231/Ser232), RIPK3, MLKL phosphorylation (Ser345), MLKL, RIPK1 phosphorylation (Ser166) protein from lung tissue sections of A. MLKL was used as loading control. (E) Quantification of protein levels in BALF of lungs from mice treated with oligomycin either control or LPS (n=5 per each group). (F) Quantification of total inflammatory cells in BALF of lungs from mice treated with oligomycin either control or LPS (n=5 per each group). Data are representative of three independent experiments. Data are mean±SD. ^*p<0.05; ^**p<0.01; ^***p<0.001 by Student’s 2-tailed t-test or ANOVA.

Figure 4. The reduced ATP5A levels in lung epithelial cells contribute to lung injury of patients with ALI.

(A) Representative images for H&E staining and (B) quantification of lung injury scores of lung tissues from patients with ARDS (ARDS) and non-ARDS subjects (Normal). Scale bars, 100 µm. (C) Representative immunofluorescence images for ATP5A staining (green) in lung epithelial cells expressing E-cadherin (red) of ARDS and Normal. Scale bars, 20 µm. DAPI-stained nuclei are shown in blue. White arrows indicate ATP5A and E-cadherin positive cells. (D) Quantification of intensity for ATP5A-positive staining and (E) quantification of ATP5A and E-cadherin-positive cells from immunofluorescence images in A. Data are representative of three independent experiments. (ARDS, n=7; Normal, n=2; n = 10 images per individual subject). Data are mean ± SD. ^**p<0.01; ^***p<0.001 by Student’s 2-tailed t-test or Mann-Whitney test.

Figure 5. The levels of RIPK3 are elevated in lung epithelial cells of patients with ARDS.

(A) Representative immunohistochemistry images for RIPK3 staining in lung tissues from patients with ARDS (ARDS) and non-ARDS subjects (Normal). Scale bars, 100 µm. Black arrows indicate RIPK3-positive cells. (B) Quantification of intensity for RIPK3-positive staining in cells and (C) quantification of RIPK3-positive cells from immunohistochemistry images in A (ARDS, n=7; Normal, n=2; n = 10 images per individual subject). (D) Representative immunoblot images of RIPK3 phosphorylation (Ser227), RIPK3, MLKL phosphorylation (Ser358), MLKL, RIPK1 phosphorylation (Ser166) protein (left) and quantification of RIPK3 phosphorylation, RIPK3, MLKL phosphorylation, RIPK1 phosphorylation protein levels (right) in lung tissues from ARDS and Normal (n=2 per each group). β-actin or MLKL were used as loading control. Data are representative of three independent experiments. Data are mean±SD. ^**p<0.01; ^***p<0.001 by Student’s 2-tailed t-test or Mann–Whitney test.

Figure 6. The activation of necroptosis by MLKL phosphorylation is elevated in lung epithelial cells of patients with ARDS.

(A) Representative immunofluorescence images for MLKL phosphorylation at Ser358 (P-MLKL) staining (green) in lung epithelial cells expressing E-cadherin (red) of lung tissues from patients with ARDS (ARDS) and non-ARDS subjects (Normal). Scale bars, 20 µm. DAPI-stained nuclei are shown in blue. (B) Quantification of intensity for P-MLKL-positive staining and (C) quantification of P-MLKL and E-cadherin-positive cells from immunofluorescence images in A (ARDS, n=7; Normal, n=2; n=10 images per individual subject). (D) Representative immunoblot images of RIPK3 phosphorylation (Ser227), RIPK3, MLKL phosphorylation (Ser358), MLKL, RIPK1 phosphorylation (Ser166) protein from lung tissue sections of A. MLKL was used as loading control. (E) A schematic diagram to summarize our new findings. Red arrow means an increase and blue arrow means a decrease in the diagram. Data are representative of 3 independent experiments. Data are mean ± SD. ^**p<0.01 by Student’s 2-tailed t-test.