Pink1-mediated mitophagy in the endothelium releases proteins encoded by mitochondrial DNA and activates neutrophil responses
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Evaluation Summary:
The authors suggest that PINK1-dependent endothelial mitophagy is pro-inflammatory by increasing the release of mitochondrial formyl peptides, one of the mitochondrial DAMPs. This study is important to identify the origin of serum formyl peptides during inflammation and to propose a new role of mitophagy in inflammation, which may be context and/or tissue specific.
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Abstract
Given their ancient evolutionary origins, eukaryotic mitochondria possess multiple vestiges of their prokaryotic ancestors. One such factor is the N-terminal formylation of proteins encoded by mitochondrial DNA. N-formylated proteins are also released by bacteria and trigger activation of immune cells such as neutrophils. Growing evidence indicate that circulating levels of mitochondrial formyl proteins are elevated in the serum of patients with excessive inflammatory responses and trigger neutrophil activation like their bacterial counterparts. However, the cellular source of these proteins, and the mechanism by which they are released into the circulation is not known. In this study, we have identified vascular endothelial cells as a source of mitophagy induced release of formyl proteins in response to inflammatory mediators in vitro. Mechanistically, endothelial mitophagy required activation of the Pink1 pathway. Using liposomal delivery of sgRNA targeting Pink1 in mice expressing endothelial-specific Cas9, we developed a mouse model in which Pink1 is specifically depleted in the endothelium. Deletion of endothelial Pink1 was remarkably protective in endotoxin-induced lung inflammation, resulting in reduced neutrophil infiltration and significantly reduced death in mice. We thus propose that endothelial cells upregulate pro-inflammatory mitophagy in response to inflammation, leading to release of mitochondrial formyl peptides and detrimental neutrophil recruitment into the lung.
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Evaluation Summary:
The authors suggest that PINK1-dependent endothelial mitophagy is pro-inflammatory by increasing the release of mitochondrial formyl peptides, one of the mitochondrial DAMPs. This study is important to identify the origin of serum formyl peptides during inflammation and to propose a new role of mitophagy in inflammation, which may be context and/or tissue specific.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 agreed to share their name with the authors.)
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Reviewer #1 (Public Review):
The authors show that LPS activates Pink1-Parkin-dependent mitophagy in lung endothelial cells, which is mediated by TNFα. Endothelial-specific Pink1-KO improves LPS-induced inflammatory responses in mice and reduces the release of one of the mitochondrial formylated proteins, ND6. These results suggest that PINK1-dependent endothelial mitophagy is pro-inflammatory and increases the release of mitochondrial formyl peptides, one of the mitochondrial DAMPs. The identification of the origin of serum formyl proteins and the elucidation of the release mechanism would be important.
The data are generally clear and convincing. The authors' conclusion would be strengthened if the authors could show that serum ND6 levels during inflammation is indeed regulated by endothelial mitophagy using their endothelial-specific …
Reviewer #1 (Public Review):
The authors show that LPS activates Pink1-Parkin-dependent mitophagy in lung endothelial cells, which is mediated by TNFα. Endothelial-specific Pink1-KO improves LPS-induced inflammatory responses in mice and reduces the release of one of the mitochondrial formylated proteins, ND6. These results suggest that PINK1-dependent endothelial mitophagy is pro-inflammatory and increases the release of mitochondrial formyl peptides, one of the mitochondrial DAMPs. The identification of the origin of serum formyl proteins and the elucidation of the release mechanism would be important.
The data are generally clear and convincing. The authors' conclusion would be strengthened if the authors could show that serum ND6 levels during inflammation is indeed regulated by endothelial mitophagy using their endothelial-specific Pink1-KO mouse model.
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Reviewer #2 (Public Review):
In this manuscript, Gajwani et al., examine activation of the PINK1 mitophagy pathway in response to bacterial lipopolysaccharide (LPS) in vasculature from mouse lung tissue, as well as proinflammatory TNF-alpha in primary human lung vascular endothelial cells.
The authors have explained their data to suggest that PINK1-dependent mitophagy can occur in response to infection within the lung vasculature, which leads to the release of formylated peptides. These peptides then stimulate proinflammatory signaling in neutrophils.
The data are intriguing as they suggest that mitophagy stimulation in endothelial cells can result in the extracellular release of mitochondrial peptides, which result in an anti-bacterial-like inflammatory response. This would a key discovery and perhaps contrasts with the brain and other …
Reviewer #2 (Public Review):
In this manuscript, Gajwani et al., examine activation of the PINK1 mitophagy pathway in response to bacterial lipopolysaccharide (LPS) in vasculature from mouse lung tissue, as well as proinflammatory TNF-alpha in primary human lung vascular endothelial cells.
The authors have explained their data to suggest that PINK1-dependent mitophagy can occur in response to infection within the lung vasculature, which leads to the release of formylated peptides. These peptides then stimulate proinflammatory signaling in neutrophils.
The data are intriguing as they suggest that mitophagy stimulation in endothelial cells can result in the extracellular release of mitochondrial peptides, which result in an anti-bacterial-like inflammatory response. This would a key discovery and perhaps contrasts with the brain and other tissues. Here, current evidence proposes that a block in mitophagy leads to accumulation of damaged mitochondria and release of mitochondrial DNA, which in turn triggers the cGAS-STING inflammatory response. These observations are important as it suggests the consequences downstream of mitophagy are context and/or tissue specific. It also indicates that any therapeutic approach will have to be tailored to specific responses.
The authors have developed useful tools and innovative approaches to monitor mitophagy signaling in cells and mice, however they need to make more use of these to robustly draw their conclusions. While the data do support the conclusions, many assumptions have been made that mean alternate explanations are possible. A consistent set of experiments across all their models would be beneficial and help strengthen this from a correlative to more mechanistic piece of work. The authors monitor mitophagy, PINK1, and an inflammatory response somewhat in isolation, with the belief that they are all linked. It is possible they are not.
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Reviewer #3 (Public Review):
Gajwani et al present an intriguing study which concludes that a key aspect of endothelial TNFa-induced inflammation involves mitochondrial clearance (mitophagy) and secretion of formylated peptides that activate neutrophils. TNFa is shown to not only increase mitophagy but also increase the secretion of mitochondrial contents, which are expected to promote enhanced inflammation. A severe mouse lung inflammation model shows that mice with reduced endothelial PINK1, a major driver of mitochondrial damage-induced mitophagy, demonstrated enhanced survival and reduced neutrophil recruitment.
This manuscript is of interest as it proposes a novel mechanism of cell-cell signaling in inflammation, involving the surprising release of mitochondrial proteins via mitophagy. This is potentially an important advance. …
Reviewer #3 (Public Review):
Gajwani et al present an intriguing study which concludes that a key aspect of endothelial TNFa-induced inflammation involves mitochondrial clearance (mitophagy) and secretion of formylated peptides that activate neutrophils. TNFa is shown to not only increase mitophagy but also increase the secretion of mitochondrial contents, which are expected to promote enhanced inflammation. A severe mouse lung inflammation model shows that mice with reduced endothelial PINK1, a major driver of mitochondrial damage-induced mitophagy, demonstrated enhanced survival and reduced neutrophil recruitment.
This manuscript is of interest as it proposes a novel mechanism of cell-cell signaling in inflammation, involving the surprising release of mitochondrial proteins via mitophagy. This is potentially an important advance. However, the results are far from conclusive. In addition to specific technical problems, they provide no evidence that this mechanism operates in vivo.
Conceptual issues.
Fig 2. In general, inflammation in endothelial cells is associated with high glycolysis, not high mitochondrial metabolism. Thus, it is important to address the question, how does TNFa trigger increased mitophagy? Is it preceded by elevated mitochondrial oxidative phosphorylation, reactive oxygen production and mitochondrial damage? Or simply mitochondrial depolarization? Is it a consequence of general upregulation of ROS? General upregulation of autophagy? Figure 3 is relevant to this question but does not answer it.
It is not clear what figures 2C,D add to the paper. Why is the occasional contact of mitochondria and mitolysosomes relevant? The absence of controls or quantification further detract from this figure.
Fig 5. What about other leukocyte populations? Is the effect of PINK1 ECKO specific to neutrophils or were they the only cells examined?
Fig 6. These experiments appear to be compromised by the presence of TNFa or FCCP in the EC conditioned medium, which could act directly on the neutrophils. Additionally, the authors provide no evidence that the effect requires PINK1.
Figure 6 also raises an important question of specificity. If the consequence of mitophagy is the release of mitochondrial content and the activation of neutrophils, wouldn't other cell types that have more mitochondrial content and more mito-phagosomal flux contribute more to neutrophil activation? Perhaps the authors could compare to other cell types and test if endothelial cells are more prone to secrete their mitochondrial content. Time courses would also improve this panel.
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