In vivo fluorescence lifetime imaging of macrophage intracellular metabolism during wound responses in zebrafish.

Veronika Miskolci,Alex J Walsh,Melissa C Skala,Michael R Lasarev,John-Demian Sauer,Jing Fan,Kelsey E Tweed,Emily C Britt,Anna Huttenlocher,Landon J Zimmerman,Courtney E Mcdougal,Mark R Cronan

doi:10.7554/elife.66080

Veronika Miskolci, Alex J Walsh + Show 10 more

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https://doi.org/10.7554/elife.66080

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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.

Highlights

Macrophages are innate immune cells that play key functions in tissue repair (Krzyszczyk et al, 2018; Wynn and Vannella, 2016)
green fluorescent protein (GFP) is suitable to image in conjunction with NAD(P)H, but it excludes the acquisition of flavin adenine dinucleotide (FAD) because they have overlapping spectra (Datta et al, 2020; Qian et al, 2021), while mCherry is compatible for simultaneous imaging with NAD(P)H and FAD (Heaster et al, 2021; Hoffmann and Ponik, 2020)
As inhibiting glycolysis reduces NADH levels (Georgakoudi and Quinn, 2012; Kolenc and Quinn, 2019), we expected the optical redox ratio to decrease in macrophages of 2-d eoxy-d-glucose (2-DG)-treated larvae compared to untreated control

Summary

Introduction

Macrophages are innate immune cells that play key functions in tissue repair (Krzyszczyk et al, 2018; Wynn and Vannella, 2016). The fluorescence intensities of NAD(P)H and FAD can be used to determine the optical redox ratio (Table 1), which provides a label-free method to monitor the oxidation-reduction state of the cell (Chance et al, 1979). We performed autofluorescence imaging of NAD(P)H and FAD to assess changes in the metabolic activity of macrophages in response to tissue damage in live zebrafish. We show that these measurements detect metabolic changes in macrophages within interstitial tissue in response to sterile damage and microbial cues with temporal and spatial resolution. We show that perturbations that modulate macrophage polarization and metabolism affect tissue repair

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