In vivo fluorescence lifetime imaging of macrophage intracellular metabolism during wound responses in zebrafish
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Curated by eLife
Evaluation Summary:
Immunometabolism is an emerging field, and to understand immune cell metabolism during inflammation and infection is of great interest. In this report, cutting edge (label free) microscopy techniques and innovative zebrafish models are used to characterize the metabolism of macrophages in situ. In the future, fluorescence microscopy approaches pioneered using zebrafish may illuminate strategies to therapeutically manipulate metabolism in human immune cells.
(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 #3 agreed to share their name with the authors.)
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Abstract
The function of macrophages in vitro is linked to their metabolic rewiring. However, macrophage metabolism remains poorly characterized in situ. Here, we used two-photon intensity and lifetime imaging of autofluorescent metabolic coenzymes, nicotinamide adenine dinucleotide (phosphate) (NAD(P)H) and flavin adenine dinucleotide (FAD), to assess the metabolism of macrophages in the wound microenvironment. Inhibiting glycolysis reduced NAD(P)H mean lifetime and made the intracellular redox state of macrophages more oxidized, as indicated by reduced optical redox ratio. We found that TNFα+ macrophages had lower NAD(P)H mean lifetime and were more oxidized compared to TNFα− macrophages. Both infection and thermal injury induced a macrophage population with a more oxidized redox state in wounded tissues. Kinetic analysis detected temporal changes in the optical redox ratio during tissue repair, revealing a shift toward a more reduced redox state over time. Metformin reduced TNFα+ wound macrophages, made intracellular redox state more reduced and improved tissue repair. By contrast, depletion of STAT6 increased TNFα+ wound macrophages, made redox state more oxidized and impaired regeneration. Our findings suggest that autofluorescence of NAD(P)H and FAD is sensitive to dynamic changes in intracellular metabolism in tissues and can be used to probe the temporal and spatial regulation of macrophage metabolism during tissue damage and repair.
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Author Response:
Reviewer #3 (Public Review):
Miskolci et al have investigated if it is possible to measure the natural fluorescence of two important co-enzymes (NADH/NADPH and FAD) in living cells to determine their metabolic status. This tests the hypothesis that changes to the relative ratio of NADH/NADPH to FAD+ reflect a shift between glycolytic and oxidative phosphorylation in living macrophages. To investigate this they have used 2-photon FLIM to measure intensity and fluorescence lifetime of NAD/NADPH and FAD+ in mouse macrophages in vitro and zebrafish macrophages in vivo in a tail injury model. By comparing their measures of NAD(P)H and FAD+ from macrophages responding to different injury or infection cues and comparing this to a maRker of inflammation (TNF-alpha) they argue that there is a reduced redox state indicative of …
Author Response:
Reviewer #3 (Public Review):
Miskolci et al have investigated if it is possible to measure the natural fluorescence of two important co-enzymes (NADH/NADPH and FAD) in living cells to determine their metabolic status. This tests the hypothesis that changes to the relative ratio of NADH/NADPH to FAD+ reflect a shift between glycolytic and oxidative phosphorylation in living macrophages. To investigate this they have used 2-photon FLIM to measure intensity and fluorescence lifetime of NAD/NADPH and FAD+ in mouse macrophages in vitro and zebrafish macrophages in vivo in a tail injury model. By comparing their measures of NAD(P)H and FAD+ from macrophages responding to different injury or infection cues and comparing this to a maRker of inflammation (TNF-alpha) they argue that there is a reduced redox state indicative of glycolytic metabolism in pro-inflammatory macrophages.
The adoption of label free imaging techniques to measure metabolic processes in cells in vivo is a valuable and important development that, although not novel to this work, will help researchers to probe cell biology in situ. FLIM using time correlated single photon counting (TCSPC) allows an accurate and robust measure of the relative state of a molecule that shows changes in its fluorescent lifetime as a consequence of changing chemical state. Although Stringari et al (doi.org/10.1038/s41598-017-03359-8) were the first to describe the utility of wavelength mixing FLIM for measuring NAD(P)H and FAD+ levels in zebrafish, they did not focus on macrophages which is the focus of this work.
The results from this work are are interesting, as they argue that it is possible to determine cell metabolism in cells within living animals without a need to use a genetically encoded sensor and they argue that pro-inflammatory macrophages in zebrafish appear to have a lower redox state, which may reflect a more glycolytic metabolism. This assumption is not tested but rather inferred based on the measures of fluorescence intensity and lifetime of endogenous NADH/NADPH and FAD coupled with a small metabolic sampling of injured tissue. This lack of corroboration for a the supposed difference in metabolism between pro-inflammatory and non-inflammatory macrophages is a weakness of the paper and makes it hard to accept the conclusion that the redox state may reflect different metabolic profiles. A biosensor for NADH/NADPH (iNap) has been demonstrated to be a sensitive tool for measuring NADPH concentration in vivo in zebrafish during the injury response (Tao et al (doi: 10.1038/nmeth.4306) and it would be intriguing to know how similar the response is of this biosensor to the label free measurements described using FLIM. This is additionally relevant as the authors also note that in mouse macrophages cultured in vitro, they observe an opposite redox response which is well supported by the literature and a variety of different methods. Why the zebrafish macrophages should show a different redox state to mouse macrophages is not clear and an alternative explanation is that the measures used do not directly reflect the metabolic profile of the cells. One further caveat to the chosen method of using fluorescence lifetime to measure the redox state of NADH/NADPH is that lifetime of NADH is affected by which proteins it is bound to. This is not accounted for in the method used for calculating the redox ratio used for defining the redox state and could potentially alter the interpretations of relative NADH/NADPH levels in a cell. The authors acknowledge this, but do not consider whether this would affect the conclusions they arrive at from their measures of NAD(P)H intensity and fluorescence lifetime in macrophages.
We thank the reviewer for their comments. We have added additional data that indicate that the imaging does indeed reflect the metabolic profile of the cells (see Metformin and STAT6 data).
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Reviewer #3 (Public Review):
Miskolci et al have investigated if it is possible to measure the natural fluorescence of two important co-enzymes (NADH/NADPH and FAD) in living cells to determine their metabolic status. This tests the hypothesis that changes to the relative ratio of NADH/NADPH to FAD+ reflect a shift between glycolytic and oxidative phosphorylation in living macrophages. To investigate this they have used 2-photon FLIM to measure intensity and fluorescence lifetime of NAD/NADPH and FAD+ in mouse macrophages in vitro and zebrafish macrophages in vivo in a tail injury model. By comparing their measures of NAD(P)H and FAD+ from macrophages responding to different injury or infection cues and comparing this to a maRker of inflammation (TNF-alpha) they argue that there is a reduced redox state indicative of glycolytic …
Reviewer #3 (Public Review):
Miskolci et al have investigated if it is possible to measure the natural fluorescence of two important co-enzymes (NADH/NADPH and FAD) in living cells to determine their metabolic status. This tests the hypothesis that changes to the relative ratio of NADH/NADPH to FAD+ reflect a shift between glycolytic and oxidative phosphorylation in living macrophages. To investigate this they have used 2-photon FLIM to measure intensity and fluorescence lifetime of NAD/NADPH and FAD+ in mouse macrophages in vitro and zebrafish macrophages in vivo in a tail injury model. By comparing their measures of NAD(P)H and FAD+ from macrophages responding to different injury or infection cues and comparing this to a maRker of inflammation (TNF-alpha) they argue that there is a reduced redox state indicative of glycolytic metabolism in pro-inflammatory macrophages.
The adoption of label free imaging techniques to measure metabolic processes in cells in vivo is a valuable and important development that, although not novel to this work, will help researchers to probe cell biology in situ. FLIM using time correlated single photon counting (TCSPC) allows an accurate and robust measure of the relative state of a molecule that shows changes in its fluorescent lifetime as a consequence of changing chemical state. Although Stringari et al (doi.org/10.1038/s41598-017-03359-8) were the first to describe the utility of wavelength mixing FLIM for measuring NAD(P)H and FAD+ levels in zebrafish, they did not focus on macrophages which is the focus of this work.
The results from this work are interesting, as they argue that it is possible to determine cell metabolism in cells within living animals without a need to use a genetically encoded sensor and they argue that pro-inflammatory macrophages in zebrafish appear to have a lower redox state, which may reflect a more glycolytic metabolism. This assumption is not tested but rather inferred based on the measures of fluorescence intensity and lifetime of endogenous NADH/NADPH and FAD coupled with a small metabolic sampling of injured tissue. This lack of corroboration for a the supposed difference in metabolism between pro-inflammatory and non-inflammatory macrophages is a weakness of the paper and makes it hard to accept the conclusion that the redox state may reflect different metabolic profiles. A biosensor for NADH/NADPH (iNap) has been demonstrated to be a sensitive tool for measuring NADPH concentration in vivo in zebrafish during the injury response (Tao et al (doi: 10.1038/nmeth.4306) and it would be intriguing to know how similar the response is of this biosensor to the label free measurements described using FLIM. This is additionally relevant as the authors also note that in mouse macrophages cultured in vitro, they observe an opposite redox response which is well supported by the literature and a variety of different methods. Why the zebrafish macrophages should show a different redox state to mouse macrophages is not clear and an alternative explanation is that the measures used do not directly reflect the metabolic profile of the cells. One further caveat to the chosen method of using fluorescence lifetime to measure the redox state of NADH/NADPH is that lifetime of NADH is affected by which proteins it is bound to. This is not accounted for in the method used for calculating the redox ratio used for defining the redox state and could potentially alter the interpretations of relative NADH/NADPH levels in a cell. The authors acknowledge this, but do not consider whether this would affect the conclusions they arrive at from their measures of NAD(P)H intensity and fluorescence lifetime in macrophages.
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Reviewer #2 (Public Review):
• The aim of this paper was to demonstrate whether FLIM-based imaging of optical redox ratio can be used to monitor metabolic states of immune cells in vivo during the course of inflammatory responses.
• The study is rigorous and well-presented and the findings are interesting and novel. The main strength is in the in vivo data, where the authors used a variety of inflammatory challenges and perturbations and were able to detect previously unreported trends in metabolic states of macrophages.
• The authors have demonstrated the potential of the technique to be used in vivo. Their initial findings are intriguing and can be followed up by more mechanistic studies.
• The work is timely, because of growing interest in the role of metabolism in immune cell signalling and functions. Relevant microscopy-based …
Reviewer #2 (Public Review):
• The aim of this paper was to demonstrate whether FLIM-based imaging of optical redox ratio can be used to monitor metabolic states of immune cells in vivo during the course of inflammatory responses.
• The study is rigorous and well-presented and the findings are interesting and novel. The main strength is in the in vivo data, where the authors used a variety of inflammatory challenges and perturbations and were able to detect previously unreported trends in metabolic states of macrophages.
• The authors have demonstrated the potential of the technique to be used in vivo. Their initial findings are intriguing and can be followed up by more mechanistic studies.
• The work is timely, because of growing interest in the role of metabolism in immune cell signalling and functions. Relevant microscopy-based assays in vivo are limited, so this innovation is important and can form the basis of further technology developments.
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Reviewer #1 (Public Review):
The zebrafish has a rich history of enabling innovative microscopy techniques, and is also a well established system to model inflammation and infection by human pathogens. Consistent with this, Miskolci et al use zebrafish to test a novel imaging approach that has great potential to significantly impact the field of immunometabolism. Fluorescence lifetime is a label-free, non-invasive imaging approach to detect metabolic changes in situ, at the level of the single cell. In this report, Miskolci et al use fluorescence lifetime imaging of NAD(P)H and FAD to detect metabolic changes in zebrafish macrophages (with temporal and spatial resolution) in response to inflammatory and infectious cues.
Miskolci et al (eLife 2019) have previously characterized inflammatory and wound healing responses to distinct caudal …
Reviewer #1 (Public Review):
The zebrafish has a rich history of enabling innovative microscopy techniques, and is also a well established system to model inflammation and infection by human pathogens. Consistent with this, Miskolci et al use zebrafish to test a novel imaging approach that has great potential to significantly impact the field of immunometabolism. Fluorescence lifetime is a label-free, non-invasive imaging approach to detect metabolic changes in situ, at the level of the single cell. In this report, Miskolci et al use fluorescence lifetime imaging of NAD(P)H and FAD to detect metabolic changes in zebrafish macrophages (with temporal and spatial resolution) in response to inflammatory and infectious cues.
Miskolci et al (eLife 2019) have previously characterized inflammatory and wound healing responses to distinct caudal fin injuries (tail wound, infection and tail wound, thermal injury). In this report, authors use these different injury models to show that fluorescence lifetime imaging can detect variations in macrophage metabolism. Although many interesting results are presented and future directions are proposed, the study in its current state is descriptive and lacks validation across different modalities. As a result, the reliability of fluorescence lifetime imaging in zebrafish macrophages is not yet convincing. Moreover, any metabolomic changes in macrophages are not functionally linked to zebrafish phenotypes (eg inflammation, bacterial burden, caudal fin regeneration).
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Evaluation Summary:
Immunometabolism is an emerging field, and to understand immune cell metabolism during inflammation and infection is of great interest. In this report, cutting edge (label free) microscopy techniques and innovative zebrafish models are used to characterize the metabolism of macrophages in situ. In the future, fluorescence microscopy approaches pioneered using zebrafish may illuminate strategies to therapeutically manipulate metabolism in human immune cells.
(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 #3 agreed to share their name with the authors.)
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