Pink1-mediated mitophagy in the endothelium releases proteins encoded by mitochondrial DNA and activates neutrophil responses during inflammation
- Department of Biochemistry and Molecular Genetics, University of Illinois, College of Medicine, United States
- Department of Oral Biology, University of Illinois, College of Dentistry, United States
- University of Illinois Cancer Center, United States
- Department of Pharmacology and Regenerative Medicine, University of Illinois, College of Medicine, United States
- Research Resources Center, University of Illinois Chicago, United States
Figures
Figure 1 with 1 supplement
Lung vascular endothelial cells initiate mitophagy in response to endotoxemic inflammation.
Mice expressing mitophagy biosensor mitoKeima (mt-Keima) were injected with Isolectin-B4 to label endothelial cells. Lungs were harvested and perfused, and mitophagy was visualized in the whole, un-sectioned lung by confocal microscopy (A). This schematic was generated using BioRender.com. A 3D mask of the endothelium was constructed and used to isolate the mt-Keima acidic/neutral ratio specifically in endothelial cells (B). Scale Bar: 10 μm. Using this method, mitophagy was measured in an endotoxemia model of inflammation. Lungs from mt-Keima mice were visualized 6 hr post-i.p. Lipopolysaccharide (LPS) injection (8 mg/kg) (C). Endothelial (D) and whole lung (E) mitophagy was measured by calculating the ratio of acidic to neutral mt-Keima (n=4 mice, 10–20 fields of view per mouse). The area of mitolysosomes was quantified as the area of the ratio above threshold. Data represent mean ± SEM. Statistical significance between PBS- and LPS-treated mice was evaluated by t-test. Source data for (D) and (E) are available in Figure 1—source data 1.
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Figure 1—source data 1
Spreadsheet containing the original source data for Figure 1D and E.
- https://cdn.elifesciences.org/articles/82205/elife-82205-fig1-data1-v2.xlsx
Figure 1—figure supplement 1
Ex vivo visualization of mt-Keima in the mouse lung.
Representative images of lungs from mt-Keima mice injected with Isolectin-B4 (IB4) to visualize the endothelium (magenta). Relative intensities of neutral, cytoplasmic mitochondria (green) and acidic, lysosomal mitochondria (red) are compared to identify regions of lower mitophagy (expanded inset a) and higher mitophagy (expanded inset b).
Figure 2 with 3 supplements
TNFα stabilizes Pink1 on the mitochondria.
Human lung microvascular endothelial cells (HLMVECs) expressing mt-Keima were treated with the inflammatory mediator TNFα (10 ng/mL), and mitophagy visualized at 3- and 6 hr post-TNFα exposure, with representative images (A). Mitophagy was calculated by measuring the ratio of acidic mitochondria to total mitochondrial area in each visual field (B). Data represent mean ± SEM from n=3 independent experiments. Statistical analysis was done using one-way ANOVA. HLMVECs were treated with TNFα for 3 hr, fixed, and immune-stained to visualize Pink1 and the mitochondrial marker Tomm20. Confocal z-stack images were analyzed for Pink1 localization relative to the mitochondria (C). Red arrows indicate areas of colocalization of Pink1 with Tomm20. Confocal images (D) were analyzed to quantify the Manders’ coefficient of overlap of Pink1 with Tomm20 (E), and the total amount of Pink1 in the mitochondrial compartment (F). Data represent mean ± SEM. Statistical significance was assessed by Welch’s t-test. Source data for (B), (E) and (F) are provided in Figure 2—source data 1.
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Figure 2—source data 1
Spreadsheet containing the original source data for Figure 2B, E and F.
- https://cdn.elifesciences.org/articles/82205/elife-82205-fig2-data1-v2.xlsx
Figure 2—figure supplement 1
TNFα induces mitochondrial depolarization in endothelial cells.
Human lung microvascular endothelial cells (HLMVECs) were treated with TNFα (10 ng/mL) for the indicated times or FCCP (1 µM, 15 min). 30 min prior to imaging, cells were stained with 20 nM tetramethylrhodamine methyl ester (TMRM). Representative images of FCCP-treated HLMVECs (A), representative images (B), and quantification of the mean TMRM fluorescence intensity ± SEM (C) of TNFα-treated HLMVECs from n=3 independent experiments are shown. Statistical analysis was done by one-way ANOVA analysis. Source data for (C) is available in Figure 2—figure supplement 1—source data 1.
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Figure 2—figure supplement 1—source data 1
Spreadsheet containing the original source data for Figure 2—figure supplement 1C.
- https://cdn.elifesciences.org/articles/82205/elife-82205-fig2-figsupp1-data1-v2.xlsx
Figure 2—figure supplement 2
Pink1 protein is stabilized by TNFα.
Representative western blot of lysates collected from human lung microvascular endothelial cells (HLMVECs) treated with TNFα for 0.5, 1, 3, 6, and 24 hr (A). Quantification of western blots representing mean ± SEM from. n=4 independent experiments (B). Statistical analysis was done by t-test. Source Data for (B) are provided in Figure 2—figure supplement 2—source data 1. Uncropped western blot images for (A) are provided in Figure 2—figure supplement 2—source data 2.
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Figure 2—figure supplement 2—source data 1
Spreadsheet containing the original source data for Figure 2—figure supplement 2B.
- https://cdn.elifesciences.org/articles/82205/elife-82205-fig2-figsupp2-data1-v2.xlsx
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Figure 2—figure supplement 2—source data 2
Uncropped western blot images for Figure 2—figure supplement 2A, with relevant bands indicated.
- https://cdn.elifesciences.org/articles/82205/elife-82205-fig2-figsupp2-data2-v2.pdf
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Figure 2—figure supplement 2—source data 3
Original western blot images for Figure 2—figure supplement 2A.
- https://cdn.elifesciences.org/articles/82205/elife-82205-fig2-figsupp2-data3-v2.zip
Figure 2—figure supplement 3
Cellular Pink1 protein levels are increased following treatment with TNFα.
Quantification of total Pink1 intensity from confocal images in Figure 2E, normalized to the total intensity of Tomm20 in each image. Data represent mean ± SEM from n=3 independent experiments. Statistical analysis performed by t-test. Source data provided in Figure 2—figure supplement 3—source data 1.
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Figure 2—figure supplement 3—source data 1
Spreadsheet containing the original source data for Figure 2—figure supplement 3.
- https://cdn.elifesciences.org/articles/82205/elife-82205-fig2-figsupp3-data1-v2.xlsx
Figure 3 with 2 supplements
Deletion of endothelial Pink1 ablates lipopolysaccharide (LPS)-induced mitophagy.
Mice were bred to express Cas9 in cells expressing Cre recombinase under a VE-Cadherin promoter, ensuring Cas9 expression specifically in endothelial cells. sgRNA against Pink1 was delivered to Cas9 expressing, or control C57BL/6 (WT) mice aged 8–12 weeks by retro-orbital i.v. injection of sgRNA containing zwitterionic vesicles. Control and Pink1 EC-KD mice were subjected to LPS (8 mg/kg body weight, i.p.) or PBS as a control. Lungs were harvested 6 hr post-treatment and analyzed for Pink1 expression via western blot (A, B). Mouse lungs were similarly prepared and harvested for immunostaining with the mitochondrial marker Tomm20, and the lysosomal marker Lamp1. DAPI was used to stain the nuclei. The proportion of mitochondria colocalized with lysosomes was calculated by creating a lysosomal mask, quantifying Tomm20 within the masked area, and dividing by total Tomm20 (C, D). Conversion of LC3B-I to LC3B-II in lung lysates of mice injected with PBS or LPS for 6 hr was quantified by western blot (E, F). Data in (B), (D), and (F) represent mean ± SEM, and statistical significance was evaluated by one-way ANOVA. Source data for (B), (D), and (F) are provided in Figure 3—source data 1. Uncropped western blot images for (A) and (E) are provided in Figure 3—source data 2.
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Figure 3—source data 1
Spreadsheet containing source data for Figure 3B, D and F.
- https://cdn.elifesciences.org/articles/82205/elife-82205-fig3-data1-v2.xlsx
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Figure 3—source data 2
Uncropped western blot images for Figure 3A and E, with relevant bands indicated.
- https://cdn.elifesciences.org/articles/82205/elife-82205-fig3-data2-v2.pdf
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Figure 3—source data 3
Original western blot images for Figure 3A and E.
- https://cdn.elifesciences.org/articles/82205/elife-82205-fig3-data3-v2.zip
Figure 3—figure supplement 1
Schematic representation of nanoparticle injection of Pink1 gRNA in Cas9-VECre mice.
Generated using BioRender.com.
Figure 3—figure supplement 2
Liposomal delivery of Pink1 sgRNA deletes Pink1 in endothelial cells, but not in non-endothelial cells.
sgRNA against Pink1 was delivered to Cas9 expressing, or control C57BL/6 mice aged 8–12 weeks by retro-orbital i.v. injection of sgRNA containing liposomes. Lungs were harvested after 24 hr and evaluated for Pink1 knockdown efficiency by western blot (A). Administration of liposomal Pink sgRNA led to an ~80% knockdown in endothelial cells but no significant knockdown in non-endothelial cells (B, C). Data represent mean ± SEM from n=3-4 mice per group. Statistical analysis was performed by t-test. Source data for (B) and (C) are provided in Figure 3—figure supplement 2—source data 1. Uncropped western blot images for (A) are available in Figure 3—figure supplement 2—source data 2.
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Figure 3—figure supplement 2—source data 1
Spreadsheet containing source data for Figure 3—figure supplement 2B and C.
- https://cdn.elifesciences.org/articles/82205/elife-82205-fig3-figsupp2-data1-v2.xlsx
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Figure 3—figure supplement 2—source data 2
Uncropped western blot images for Figure 3—figure supplement 2A, with relevant bands indicated.
- https://cdn.elifesciences.org/articles/82205/elife-82205-fig3-figsupp2-data2-v2.pdf
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Figure 3—figure supplement 2—source data 3
Original western blot images for Figure 3—figure supplement 2A.
- https://cdn.elifesciences.org/articles/82205/elife-82205-fig3-figsupp2-data3-v2.zip
Figure 4 with 3 supplements
Endothelial Pink1 sensitizes mice to lipopolysaccharide (LPS).
Control and Pink1 EC-KD mice were injected with LPS (10 mg/kg body weight), and survival was monitored over 7 days (A). n=20 (10 male and 10 female) mice per group. Statistical analysis was performed using log rank (Mantel-Cox) test. To study neutrophil infiltration and activation, control (WT) and endothelial-specific Pink1 knockdown (Pink1 EC-KD) mice were injected with LPS (8 mg/kg body weight). 6- and 24 hr later, lungs were perfused and harvested and analyzed for the number of infiltrated Ly6G+neutrophils by flow cytometry (B, C). Data are presented as mean ± SEM. Neutrophil activation was measured by CD11b expression on Ly6G + cells (D, E). Data represent median ± SEM from n=3–6 mice per group. IL-1β levels in the whole lung after 6 hr LPS treatment were measured by western blot (F, G). n=4 mice for PBS, and n=6 mice for LPS-treated groups, with data represented as mean ± SEM. Statistical significance for (B–E) and (G) was determined by one-way ANOVA. Source Data for (B), (C), (D), (E), and (F) are provided in Figure 4—source data 1 . Uncropped western blot images for (F) are available in Figure 4—source data 2.
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Figure 4—source data 1
Spreadsheet containing source data for Figure 4A, B, C, D, E and G.
- https://cdn.elifesciences.org/articles/82205/elife-82205-fig4-data1-v2.xlsx
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Figure 4—source data 2
Uncropped western blot images for Figure 4F, with relevant bands indicated.
- https://cdn.elifesciences.org/articles/82205/elife-82205-fig4-data2-v2.pdf
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Figure 4—source data 3
Original western blot images for Figure 4F.
- https://cdn.elifesciences.org/articles/82205/elife-82205-fig4-data3-v2.zip
Figure 4—figure supplement 1
Endothelial Pink1 does not alter lung permeability.
Control and Pink1 EC-KD mice were injected with lipopolysaccharide (LPS; 8 mg/kg), or PBS as a control. Mice were injected with Evans Blue Albumin (EBA), and lungs perfused and harvested 12–14 hr post-injection. EBA in the lungs was quantified (A). Data represent mean ± SEM from n=3–4 mice per group. Statistical analysis was performed using one-way ANOVA. VE-Cadherin levels were measured in lungs 6 hr following LPS injection by western blot (B). β-Actin was measured as a loading control. n=2–3 mice per group. Source Data for (A) is provided in Figure 4—figure supplement 1—source data 1. Uncropped western blot images for (B) are available in Figure 4—figure supplement 1—source data 2.
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Figure 4—figure supplement 1—source data 1
Spreadsheet containing source data for Figure 4—figure supplement 1A.
- https://cdn.elifesciences.org/articles/82205/elife-82205-fig4-figsupp1-data1-v2.xlsx
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Figure 4—figure supplement 1—source data 2
Uncropped western blot images for Figure 4—figure supplement 1B, with relevant bands indicated.
- https://cdn.elifesciences.org/articles/82205/elife-82205-fig4-figsupp1-data2-v2.pdf
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Figure 4—figure supplement 1—source data 3
Original western blot images for Figure 4—figure supplement 1B.
- https://cdn.elifesciences.org/articles/82205/elife-82205-fig4-figsupp1-data3-v2.zip
Figure 4—figure supplement 2
Endothelial Pink1 does not regulate lung edema.
Control and Pink1 EC-KD mice were injected with lipopolysaccharide (LPS; 8 mg/kg), or PBS as a control. 6 hr following lipopolysaccharide (LPS) injection, lungs were harvested and weighed. Following drying at 60 °C, lungs were weighed again, and the wet-to-dry ratio was calculated as a measure of lung edema. Data represent mean ± SEM from n=5 mice per group. Statistical significance was measured by Welch’s t-test. Source data are provided in Figure 4—figure supplement 2—source data 1.
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Figure 4—figure supplement 2—source data 1
Spreadsheet containing source data for Figure 4—figure supplement 2.
- https://cdn.elifesciences.org/articles/82205/elife-82205-fig4-figsupp2-data1-v2.xlsx
Figure 4—figure supplement 3
Endothelial Pink1 does not alter endothelial ICAM expression following lipopolysaccharide (LPS) treatment.
Control and Pink1 EC-KD mice were injected with LPS (8 mg/kg). 6- (A) and 24- (B) hr later, lungs were perfused and harvested and analyzed for ICAM expression in CD31+endothelial cells by flow cytometry. Data are presented as median ± SEM from n=3–5 mice per LPS-treated group. Statistical significance was evaluated by one-way ANOVA. Source data are provided in Figure 4—figure supplement 3—source data 1.
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Figure 4—figure supplement 3—source data 1
Spreadsheet containing source data for Figure 4—figure supplement 3A, B.
- https://cdn.elifesciences.org/articles/82205/elife-82205-fig4-figsupp3-data1-v2.xlsx
Figure 5 with 1 supplement
Endothelial cells release ND6 in response to inflammation.
Human lung microvascular endothelial cells (HLMVECs) were treated with TNFα for 1 hr, washed, and incubated in low serum media for 24 hr. Conditioned cell culture media was collected and filtered and placed into the lower chamber of a trans-well assay plate. Differentiated HL-60 cells (dHL-60) were placed in the upper chamber. Transmigration in response to media from TNFα-treated and control cells was analyzed (A). Data are presented as mean ± SEM from n=3 independent experiments. dHL-60 cells were resuspended in HLMVEC conditioned media, incubated for 10 min, lysed, and analyzed for Erk phosphorylation by western blot (B, C). Data represent mean ± SEM from n=3 independent experiments. HLMVECs were treated with TNFα for 24 hr. Conditioned media was then collected and analyzed for the presence of ND6 by ELISA (D). Data represent mean ± SEM from n=3 independent experiments. Statistical significance for (A), (C), and (D) was analyzed by t-test. C57BL/6 mice were injected with lipopolysaccharide (LPS; 8 mg/kg, i.p.), and blood plasma collected after 0, 3, 6, and 24 hr. Plasma proteins were precipitated using trichloroacetic acid (TCA, 13%), resuspended in Laemmli buffer, and analyzed by western blot (E, F). WT and Pink1 EC-KD mice were injected with LPS, and plasma collected after 6 hr for TCA precipitation and analysis by western blot (G, H). Statistical analysis for graphs (F) and (H) was performed using one-way ANOVA. Original Data for (A), (C), (D), (F), and (H) available in , Figure 5—source data 1. Uncropped western blot images for (B), (E), and (G) provided in Figure 5—source data 2.
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Figure 5—source data 1
Spreadsheet containing source data for Figure 5A, C, D, F and H.
- https://cdn.elifesciences.org/articles/82205/elife-82205-fig5-data1-v2.xlsx
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Figure 5—source data 2
Uncropped western blot images for Figure 5B, E and G, with relevant bands indicated.
- https://cdn.elifesciences.org/articles/82205/elife-82205-fig5-data2-v2.pdf
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Figure 5—source data 3
Original western blot images for Figure 5B, E and G.
- https://cdn.elifesciences.org/articles/82205/elife-82205-fig5-data3-v2.zip
Figure 5—figure supplement 1
Mitochondrial formylated peptide induces trans-well migration of dHL-60 cells.
Purified mitochondrial formyl peptide (fMIT, 100 nM) was added to the lower chamber of a trans-well assay plate. Differentiated HL-60 cells (dHL-60) were placed in the upper chamber. Transmigration in response to media from TNFα-treated and control cells was quantified (A). Statistical analysis was performed using Welch’s t-test. dHL-60 cells were exposed to 10 nM fMLP or fMIT for 10 min, lysed, and evaluated for Erk phosphorylation by western blot (B, C).Data in graphs are presented as mean ± SEM, and statistical significance was assessed by one-way ANOVA. Source data for (A) and (C) are available in Figure 5—figure supplement 1—source data 1. Uncropped western blot images for (B) are provided in Figure 5—figure supplement 1—source data 2.
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Figure 5—figure supplement 1—source data 1
Spreadsheet containing source data for Figure 5—figure supplement 1A and C.
- https://cdn.elifesciences.org/articles/82205/elife-82205-fig5-figsupp1-data1-v2.xlsx
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Figure 5—figure supplement 1—source data 2
Uncropped western blot images for Figure 5—figure supplement 1B, with relevant bands indicated.
- https://cdn.elifesciences.org/articles/82205/elife-82205-fig5-figsupp1-data2-v2.pdf
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Figure 5—figure supplement 1—source data 3
Original western blot images for Figure 5—figure supplement 1B.
- https://cdn.elifesciences.org/articles/82205/elife-82205-fig5-figsupp1-data3-v2.zip
Figure 6
Inflammation-induced endothelial Pink1 activity releases formylated proteins to enhance inflammation.
In response to inflammatory stimulus, Pink1 is activated in endothelial cells, leading to mitophagy and release of mitochondrial proteins such as ND6, which contain a formylated methionine (fMet) at the N-terminus. Bacteria, which share a prokaryotic ancestor with mitochondria, also produce and release N-formyl proteins. Both mitochondrial and bacterial N-formyl proteins activate neutrophils through formyl peptide receptors (FPRs), leading to increased Erk phosphorylation and increased neutrophil recruitment. Excessive neutrophil recruitment leads to increased aberrant inflammation. This schematic was created using BioRender.com.
Author response image 1
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Pink1-mediated mitophagy in the endothelium releases proteins encoded by mitochondrial DNA and activates neutrophil responses during inflammation
eLife 15:e82205.
https://doi.org/10.7554/eLife.82205
