Editor's evaluation: In vivo fluorescence lifetime imaging of macrophage intracellular metabolism during wound responses in zebrafish

Abstract

Article Figures and data Abstract Editor's evaluation Introduction Results Discussion Materials and methods Data availability References Decision letter Author response Article and author information 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. Editor's evaluation Immunometabolism is an emerging field, and to understand immune cell metabolism during inflammation and infection is of great interest. In this report, cutting edge 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. https://doi.org/10.7554/eLife.66080.sa0 Decision letter Reviews on Sciety eLife's review process Introduction Macrophages are innate immune cells that play key functions in tissue repair (Krzyszczyk et al., 2018; Wynn and Vannella, 2016). The heterogeneity and diversity of macrophage phenotypes and functions are well documented in vitro (Martinez and Gordon, 2014; Mills et al., 2014; Murray, 2017). However, there is a gap in understanding macrophage phenotypes in interstitial tissues during tissue damage and repair. This is particularly important because distinct macrophage populations play important roles in wound healing and tissue regeneration. Macrophages are commonly described as classically (M1) or alternatively (M2) activated, with both subsets playing critical roles in wound healing (Krzyszczyk et al., 2018; Wynn and Vannella, 2016). The M1/M2 classification, especially in the context of in vivo biology, is controversial (Murray et al., 2014; Orecchioni et al., 2019), and more likely represents a continuum of activation states. The importance of metabolic regulation of macrophage function was not appreciated until more recently, when it was recognized that some metabolic pathways are profoundly altered in classically activated macrophages (Jha et al., 2015; Tannahill et al., 2013). For example, classically activated macrophages are glycolytic, while oxidative phosphorylation is the main fuel source during alternative activation in vitro (O’Neill et al., 2016; Van den Bossche et al., 2017). This recent progress has led to the emergence of metabolic reprogramming as a hallmark of immune cell activation, and supports the premise that the metabolic state is not an outcome but rather a determinant of immune cell activation and function (O’Neill and Pearce, 2016; Ryan and O’Neill, 2020). While it is well documented that macrophages exhibit plasticity as wounds repair and convert from M1 to M2 over the course of wound healing (Krzyszczyk et al., 2018), the metabolic regulation of macrophage function within interstitial tissue during wound repair remains unclear (Caputa et al., 2019). We need additional tools to detect the polarization and metabolic phenotypes of macrophages within interstitial tissues in live animals. Autofluorescence imaging of the intensities and lifetimes of metabolic coenzymes is an attractive method to monitor macrophage metabolism and function in vivo. The reduced forms of nicotinamide adenine dinucleotide (phosphate) (NAD(P)H) and oxidized flavin adenine dinucleotide (FAD) are endogenous metabolic coenzymes that are autofluorescent. 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). Multiple definitions of the optical redox ratio exist, but here we use NAD(P)H/(NAD(P)H + FAD), since an increase in the optical redox ratio intuitively corresponds with a more reduced intracellular environment, suggestive of an increase in glycolysis, and it normalizes the values to be between 0 and 1 (Walsh et al., 2021). The fluorescence lifetime measures the time a molecule spends in the excited state before decaying back to the ground state. The fluorescence lifetimes of NAD(P)H and FAD are distinct in their free and protein-bound states, which provide a label-free measurement of their enzyme-binding activities (Table 1; Georgakoudi and Quinn, 2012; Kolenc and Quinn, 2019). Fluorescence lifetime imaging microscopy (FLIM) has several advantages over intensity measurements, because FLIM provides additional biological information by distinguishing the protein-bound and free states, and is not dependent on the cellular concentrations of the coenzymes (Datta et al., 2020; Walsh and Skala, 2015). Importantly, FLIM is a label-free and noninvasive method to detect metabolic changes in situ and can also resolve metabolic heterogeneity within a cell population (Heaster et al., 2019; Sharick et al., 2019; Walsh et al., 2021; Walsh and Skala, 2015). Table 1 Definition of autofluorescence imaging endpoints. EndpointsDefinitionInterpretationOptical redox ratioChance et al., 1979INAD(P)HINAD(P)H+IFADIncrease in optical redox ratio (ORR) = more reduced intracellular environment; likely increase in glycolysis; decrease in ORR = more oxidized intracellular environment; likely decrease in glycolysis. (I, intensity)Nicotinamide adenine dinucleotide (phosphate) (NAD(P)H) Short lifetime of NAD(P)HFree/unbound NAD(P)HNAD(P)H τ2Long lifetime of NAD(P)HNAD(P)H bound to a proteinNAD(P)H α1Fractional component of free NAD(P)Hα2 is fractional component of bound NAD(P)H, α1+α2=1; quantifies the pools of NAD(P)H in free and bound statesNAD(P)H mean lifetime ()τm=τ1α1+τ2α2Weighted average of individual lifetime endpoints (); one can look at changes in individual endpoints to see what drives changes in τm; for instance, a decrease in τm can be due to increase in α1, decrease in τ1, and/or decrease in τ2Flavin adenine dinucleotide (FAD) τ1Short lifetime of FADFAD bound to a proteinFAD τ2Long lifetime of FADFree/unbound FADFAD α1Fractional component of bound FADα2 is fractional component of free FAD, α1+α2=1; quantifies the pools of FAD in free and bound statesFAD τmτm=τ1α1+τ2α2Weighted average of individual lifetime endpoints (τ1,τ2,α1,α2); one can look at changes in individual endpoints to see what drives changes in τm; for instance, a decrease in τm can be due to increase in α1, decrease in τ1, and/or decrease in τ2Optical Metabolic Imaging (OMI) indexWalsh and Skala, 2015ORRi<ORR>+NAD(P)Hτmi<NAD(P)Hτm>−FADτmi<FADτm>Composite measure of mean-centered optical redox ratio and mean lifetimes of NAD(P)H and FAD; increase in the OMI index corresponds to increased redox ratio, and increased NAD(P)H and FAD protein-binding activities Zebrafish represents a powerful system to study macrophage polarization and tissue repair. Live imaging has revealed the presence of both M1 (TNFα+) and M2 (TNFα−) macrophages in wounded tissues (Miskolci et al., 2019; Nguyen-Chi et al., 2017; Nguyen-Chi et al., 2015). Here, 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 also show that perturbations that modulate macrophage polarization and metabolism affect tissue repair. Results Autofluorescence imaging detects oxidized intracellular redox state in macrophages in vivo upon 2-deoxy-d-glucose treatment To determine if a known glycolysis inhibitor alters macrophage metabolism in vivo, we imaged NAD(P)H and FAD in wounded zebrafish larvae in the absence and presence of 2-deoxy-d-glucose (2-DG). 2-DG is a glucose analog and acts as a competitive inhibitor of glycolysis at the step of phosphorylation of glucose by hexokinase (Pelicano et al., 2006). To isolate autofluorescence signals associated with macrophages from the whole tissue, we used mCherry and green fluorescent protein (GFP) transgenic reporter lines. GFP is suitable to image in conjunction with NAD(P)H, but it excludes the acquisition of 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). The traditional serial acquisition of NAD(P)H and FAD was not suitable for imaging motile cells, such as macrophages, in live larvae. To accommodate cell movement during image acquisition in live larvae, we employed wavelength mixing that allows for simultaneous acquisition in three different channels (Stringari et al., 2017). We performed simple tail fin transection on transgenic larvae (Tg(mpeg1:mCherry-CAAX) that labels the plasma membrane of macrophages with mCherry), and performed autofluorescence imaging of NAD(P)H and FAD at the wound region (Figure 1A) at 3–6 hr post tail transection (hptt) in the absence or presence of 2-DG. 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-DG-treated larvae compared to untreated control. Indeed, the optical redox ratio was significantly lower in macrophages in the 2-DG-treated larvae (Figure 1B and C). This change was driven by a decrease in NAD(P)H intensity in treated larvae, while FAD intensity remained similar to control levels (data not shown). Inhibition of glycolysis was associated with a significant reduction of the mean lifetime (τm) of NAD(P)H in macrophages, with only a marginal reduction in FAD τm (Figure 1D and E). We also observed significant reduction for NAD(P)H and FAD τ2, and increase for NAD(P)H α1 (Figure 1—figure supplement 1A-F). These effects on NAD(P)H and FAD lifetime endpoints were similar to the effects observed with 2-DG treatment of activated T cells (Walsh et al., 2021). In sum, macrophages were more oxidized following treatment with a glycolysis inhibitor, and these findings support the utility of using autofluorescence imaging of metabolic coenzymes to detect changes in metabolic activity of macrophages in situ. Figure 1 with 1 supplement see all Download asset Open asset Inhibition of glycolysis reduces the optical redox ratio of macrophages following simple transection. Tail fin transection distal to the notochord was performed using transgenic zebrafish larvae (Tg(mpeg1:mCherry-CAAX) that labels macrophages in the plasma membrane with mCherry) at 3 days post fertilization, and autofluorescence imaging of nicotinamide adenine dinucleotide (phosphate) (NAD(P)H) and flavin adenine dinucleotide (FAD) was performed on live larvae at 3–6 hr post tail transection (hptt) that were either untreated (control) or treated with 5 mM 2-deoxy-d-glucose (2-DG) (glycolysis inhibitor) for 1 hr prior to imaging. (A) Schematic showing the area where wounding (black line) and imaging (blue box) were performed. (B) Representative images of mCherry (to show macrophages), optical redox ratio, and NAD(P)H and FAD mean lifetimes (τm) are shown; macrophages in mCherry channel were outlined with dashed lines and the area was overlaid in the optical redox ratio and lifetime images to show corresponding location; scale bar = 50 µm. Quantitative analysis of (C) optical redox ratio, (D) NAD(P)H and (E) FAD mean lifetimes (τm) from two biological repeats (control = 90 cells/9 larvae, 2-DG = 123 cells/9 larvae) is shown; quantitative analysis of associated individual lifetime endpoints (τ1,τ2,α1) and sample size for each repeat are included in Figure 1—figure supplement 1. The optical redox ratio and τm were log transformed prior to analysis. p values represent statistical analysis of the overall effects. Estimated means with 95% CI and overall effects with p values are included in Figure 1—source data 1. Figure 1—source data 1 Related to Figure 1. https://cdn.elifesciences.org/articles/66080/elife-66080-fig1-data1-v1.xlsx Download elife-66080-fig1-data1-v1.xlsx Autofluorescence imaging detects metabolic changes in macrophages at the infected tail wound To determine if autofluorescence imaging could distinguish different macrophage populations in a whole organism, we used a zebrafish Listeria monocytogenes (Lm)-infected tail wound model (Miskolci et al., 2019). This infection induces the recruitment of M1 macrophages as detected by a high level of TNFα expression (Miskolci et al., 2019). In contrast, most macrophages are devoid of TNFα expression following a simple transection (Miskolci et al., 2019), and likely represent a differentially activated M2-like population (Nguyen-Chi et al., 2015). Based on the differential activation profiles, we hypothesized that we would detect differences in the metabolic activity of macrophages at the simple and Lm-infected transection wounds. We performed tail fin transection in the absence or presence of Lm on double transgenic (Tg(tnf:GFP) × Tg(mpeg1:mCherry-CAAX)) larvae and performed autofluorescence imaging of NAD(P)H at the wound region on live larvae at 48 hr post wound (hpw). 48 hpw was chosen as a representative timepoint when the proportion of TNFα+ cells at these wounds was sufficiently different between the two wound models; most macrophages (~70%) are TNFα− in response to simple transection, whereas most macrophages (~80%) are TNFα+ at the infected wound, and these proportions do not significantly change at later timepoints (Miskolci et al., 2019). We performed autofluorescence imaging in conjunction with the TNFα reporter line (tnf:GFP) in order to monitor and group macrophages by TNFα expression during image analysis. The TNFα reporter line relies on GFP expression to report transcriptional activity of tnfα (Marjoram et al., 2015), which precludes acquisition of FAD measurements. As a result, in this experiment we were not able to monitor changes in the intracellular optical redox ratio. Macrophages at the wound region were identified based on plasma membrane-localized mCherry expression as above. The infected tail wound recruited significantly more macrophages compared to the uninfected control (simple transection), and most macrophages at the infected tail wound expressed high levels of TNFα, while the majority lacked TNFα expression at the uninfected control wounds (Figure 2A, Figure 2—figure supplement 1D), consistent with our previous report (Miskolci et al., 2019). We detected a significant reduction in the mean lifetime (τm) of NAD(P)H for TNFα+ macrophages relative to TNFα− macrophages in both the uninfected control and Lm-infected tail wounds (Figure 2B). While both types of wounds showed similar trends for TNFα+ and TNFα− macrophages, NAD(P)H τm was further reduced in macrophages from Lm-infected tail wounds relative to uninfected control when comparing either the TNFα− or TNFα+ groups (Figure 2B). Similar trends were observed for the individual lifetime components (τ1,τ2) of NAD(P)H as those observed for τm (Figure 2—figure supplement 1A,B), while we did not detect any significant changes in the fractional component of free NAD(P)H (α1) in any of the comparisons (Figure 1—figure supplement 1C). Figure 2 with 1 supplement see all Download asset Open asset Mean lifetime of nicotinamide adenine dinucleotide (phosphate) (NAD(P)H) is reduced in TNFα+ macrophages at the infected tail wound. Tail fin transection distal to the notochord was performed using N-phenylthiourea-treated double transgenic zebrafish larvae (Tg(tnf:GFP) x Tg(mpeg1:mCherry-CAAX), a TNFα reporter line in combination with a line that labels macrophages in the plasma membrane with mCherry) at 3 days post fertilization in the absence or presence of Listeria monocytogenes (Lm). Autofluorescence imaging of NAD(P)H was performed on live larvae at 48 hr post wound (Figure 1A). (A) Representative images of mCherry expression to show macrophages, GFP to show TNFα expression, and NAD(P)H mean lifetime (τm) are shown for control or Lm-infected tail wounds. Macrophages in the mCherry channel were outlined with dashed lines and the area was overlaid in GFP and lifetime images to show corresponding location; in the infected condition only a few macrophages are outlined as examples; scale bar = 50 µm. (B) Quantitative analysis of NAD(P)H mean lifetime (τm) from three biological repeats (control TNFα− = 184 cells/16 larvae, control TNFα+ = 75 cells/16 larvae, infected TNFα− = 258 cells/16 larvae, infected TNFα+ = 789 cells/16 larvae) is shown; quantitative analysis of associated individual lifetime endpoints (τ1,τ2,α1) and sample size for each repeat are included in Figure 2—figure supplement 1. The τm was log transformed prior to analysis. Interaction between treatment and GFP expression was included to analyze whether either factor modified the effect of the other; no interaction was found. p values represent statistical analysis of the overall effects. Estimated means with 95% CI and overall effects with p values are included in Figure 2—source data 1. Figure 2—source data 1 Related to Figure 2. https://cdn.elifesciences.org/articles/66080/elife-66080-fig2-data1-v1.xlsx Download elife-66080-fig2-data1-v1.xlsx Next, we repeated the same set of experiments but without the TNFα reporter, to acquire FAD measurements in order to monitor changes in the optical redox ratio. NAD(P)H τm, τ1 and τ2 were significantly reduced in macrophages at the Lm-infected wound (Figure 3D, Figure 3—figure supplement 1A,B), consistent with the measurements above (Figure 2B, Figure 2—figure supplement 1A,B). We found that NAD(P)H α1 significantly increased in macrophages at the Lm-infected wound (Figure 3—figure supplement 1C). The presence of infection at the tail wound did not induce any significant changes in FAD lifetime endpoints (Figure 3—figure supplement 1D-G). Interestingly, the optical redox ratio was significantly reduced in macrophages at the highly inflammatory Lm-infected wound compared to the uninfected control, indicating that TNFα+ (M1-like) macrophage population is more oxidized compared to TNFα− (M2-like) in vivo (Figure 3A and B). The Optical Metabolic Imaging (OMI) index, a composite measure of the optical redox ratio, NAD(P)H τm and FAD τm (Table 1), was also lower in macrophages at the Lm-infected wound (Figure 3C). These findings were unexpected considering the observed increase of the optical redox ratio in the context of in vitro infection of bone marrow-derived macrophages (BMDM) with Lm (Figure 3—figure supplement 1I,J), indicating a more reduced intracellular redox state, consistent with previous publications of Listeria infection of macrophages in vitro (Gillmaier et al., 2012). We reasoned this result may be influenced by the presence of an intracellular pathogen in macrophages, and not solely due to a more proinflammatory macrophage phenotype. To test this, we next measured changes in the intracellular metabolism of macrophages in the context of thermal injury that also induces a TNFα+ macrophage population, but in the absence of infection. Figure 3 with 1 supplement see all Download asset Open asset Optical redox ratio and mean lifetime of nicotinamide adenine dinucleotide (phosphate) (NAD(P)H) are reduced in macrophages at the infected tail wound. Tail fin transection distal to the notochord was performed using transgenic zebrafish larvae (Tg(mpeg1:mCherry-CAAX) that labels macrophages in the plasma membrane with mCherry) at 3 days post fertilization in the absence or presence of Listeria monocytogenes (Lm). Autofluorescence imaging of NAD(P)H and flavin adenine dinucleotide (FAD) was performed on live larvae at 48 hr post wound (Figure 1A). (A) Representative images of mCherry expression to show macrophages, optical redox ratio, and NAD(P)H and FAD mean lifetimes (τm) are shown for control or infected tail wounds; macrophages were outlined with dashed lines and the area was overlaid in the optical redox ratio and lifetime images to show corresponding area; in the infected condition only a few macrophages are outlined as examples; scale bar = 50 µm. Quantitative analysis of (B) optical redox ratio, (C) Optical Metabolic Imaging index, and (D) NAD(P)H mean lifetime (τm) from three biological repeats (control = 105 cells/16 larvae, infected = 761 cells/14 larvae) is shown; quantitative analysis of associated NAD(P)H and FAD mean (τm) and individual lifetime endpoints (τ1,τ2,α1), and sample size for each repeat are included in Figure 3—figure supplement 1. p values represent statistical analysis of the overall effects. Estimated means with 95% CI and overall effects with p values are included in Figure 3—source data 1. Figure 3—source data 1 Related to Figure 3. https://cdn.elifesciences.org/articles/66080/elife-66080-fig3-data1-v1.xlsx Download elife-66080-fig3-data1-v1.xlsx Autofluorescence imaging resolves changes in the metabolic activity of macrophages over the course of thermal tissue damage To measure metabolic activity of macrophages during robust tissue damage, we used our zebrafish thermal injury tail wound model (LeBert et al., 2018). The burn wound elicits the recruitment of an M1-like macrophage population, as detected by TNFα expression (Miskolci et al., 2019). Unlike at the infected wound where TNFα expression in macrophages persists, TNFα+ macrophages peak at 24 hpw and resolve thereafter, as most macrophages at the wound are TNFα− by 72 hpw following thermal injury (Miskolci et al., 2019). We chose these two timepoints to compare the metabolic activity of macrophages in response to simple transection and thermal injury. Since macrophages are mostly TNFα− throughout the course of the wound response following a simple transection, we hypothesized that the metabolic activity of macrophages would be different at 24 hpw, but similar at 72 hpw, when comparing the two wound models. We performed tail transection or generated a burn wound distal to the notochord on transgenic (Tg(mpeg1:mCherry-CAAX)) larvae, and performed autofluorescence imaging at the wound region on live larvae at 24 and 72 hpw. As expected, we observed significant differences in the metabolic activity of macrophages between the wounds at 24 hpw, but the cellular metabolism was similar at 72 hpw. Importantly, macrophages at the burn wound had a more oxidized redox state relative to macrophages at the simple transection at 24 hpw, indicated by the lower optical redox ratio and OMI index (Figure 4A–C). In addition, NAD(P)H τm and τ1 were lower, while α1 was higher in macrophages at the burn compared to simple transection at 24 hpw (Figure 4D, Figure 4—figure supplement 1A,C); we did not detect any significant changes in FAD lifetime endpoints at 24 hpw (Figure 4—figure supplement 1D-G). Macrophages are mostly TNFα− at both wound types by 72 hpw (Miskolci et al., 2019), suggesting that the macrophage populations present at these wounds have similar activation states and are thereby likely to have similar metabolic activity. Accordingly, the optical redox ratio and OMI index were not different between macrophages of the simple transection and burn wound at 72 hpw (Figure 4B and C). The mean lifetime of NAD(P)H was significantly lower in macrophages at the burn wound relative to the simple transection at 72 hpw (Figure 4D), while it was similar for FAD (Figure 4—figure supplement 1D). Most of the individual lifetime endpoints (τ1,τ2 and α1) of NAD(P)H and FAD were also similar between the two wounds at 72 hpw (Figure 4—figure supplement 1A-G). Furthermore, we also detected temporal changes in the metabolic activity of macrophages during wound responses. The optical redox ratio and OMI index of macrophages increased over time at both wound types (Figure 4B and C), indicating a more reduced redox state. This would be expected as TNFα+ macrophages resolve at both wound types over time (Miskolci et al., 2019). In line with this, NAD(P)H τm,τ1 and τ2 increased, while α1 decreased at both wound types over time (Figure 4D, Figure 4—figure supplement 1A-C). We also detected time-related changes in FAD endpoints; FAD τm, and τ1 decreased over time at both wounds (Figure 4—figure supplement 1D,E). Collectively, we found that TNFα+ macrophage population was more oxidized, as indicated by a decrease in the redox ratio, and was associated with a decrease in NAD(P)H mean lifetime relative to a TNFα− macrophage population in context of both infected and sterile injury (Table 2). Table 2 Summary of changes in optical redox ratio, Optical Metabolic Imaging (OMI) index, and nicotinamide adenine dinucleotide (phosphate) (NAD(P)H) lifetime endpoints. Changes in treated samples are shown relative to the control. Control, simple tail transection (uninfected); nd, not different. Control versus2-Deoxy-d-glucoseLm-infected wound TNFα−Lm-infected wound TNFα+Lm-infected woundBurn wound24 hrBurn wound72 hrOptical redox ratio↓--↓↓ndOMI index---↓↓ndNAD(P)H τm↓↓↓↓↓↓NAD(P)H τ1nd↓↓↓↓ndNAD(P)H τ2↓↓↓↓ndndNAD(P)H α1↑ndnd↑↑↑ Figure 4 with 1 supplement see all Download asset Open asset Autofluorescence imaging resolves temporal changes in the metabolic activity of macrophages at sterile tail wounds. Tail fin transection (Tt) or thermal injury (burn) distal to the notochord was performed using transgenic zebrafish larvae (Tg(mpeg1:mCherry-CAAX) that labels macrophages in the plasma membrane with mCherry) at 3 days post fertilization. Autofluorescence imaging of nicotinamide adenine dinucleotide (phosphate) (NAD(P)H) and flavin adenine dinucleotide (FAD) was performed on live larvae at 24 and 72 hr post wound (Figure 1A). (A) Representative images of mCherry expression to show macrophages, optical redox ratio, and NAD(P)H and FAD mean lifetimes (τm) are shown for Tt or burn wounds; macrophages were outlined with dashed lines and the area was overlaid in the optical redox ratio and lifetime images to show corresponding location of macrophages; scale bar = 50 µm. Quantitative analysis of (B) optical redox ratio, (C) Optical Metabolic Imaging index, and (D) NAD(P)H mean lifetime (τm) from three biological repeats (Tt-24 hr = 322 cells/16 larvae, burn-24 hr = 850 cells/14 larvae, Tt-72 hr = 213 cells/12 larvae, burn-72 hr = 578 cells/11 larvae) is shown; quantitative analysis of associated NAD(P)H and FAD mean (τm) and individual lifetime endpoints (τ1,τ2,α1), and sample size for each repeat are included in Figure 4—figure supplement 1. Interaction between treatment and time was included to analyze whether either factor modified the effect of the other; strong interaction was detected for the optical redox ratio. p values represent statistical analysis of the overall effects. Estimated means with 95% CI and overall effects with p values are included in Figure 4—source data 1. (E) Tail fin tissue was collected distal to the caudal vein/artery loop (blue box) 24 hr following either tail transection or thermal injury distal to the notochord (black line) for mass spec analysis of small metabolites to compare the global trend of changes in redox metabolites with that measured by autofluorescence imaging; metabolomics data shown in (E) and (F) are from four biological repeats. (F) Metabolite abundance measured by either autofluorescence imaging or mass spec in transection sample was normalized by that in burn or (G) was used to calculate the redox ratio in transection (Tt) or burn samples. We included NADPH abundance in the redox ratio calculated using mass spec measurements. *NADPH and NADH intensities could not be collected separately by autofluorescence imaging as their fluorescence spectra overlap, thereby were measured collectively. Figure 4—source data 1 Related to Figure 4. https://cdn.elifesciences.org/articles/66080/elife-66080-fig4-data1-v1.xlsx Download elife-66080-fig4-data1-v1.xlsx To substantiate the metabolic changes observed by autofluorescence imaging in macrophages at the sterile tail wounds, we tested if we would see similar changes in tail fin tissue using targeted liquid chromatography-mass spectrometry (LS-MS)-based method to analyze the abundance of NADH, NAD(P)H, and FAD. We performed simple transection or burn wound distal to the notochord on unlabeled wild-type zebrafish larvae and collected the tail fin tissue distal to the caudal vein/artery loop (to remain close to the wound microenvironment) 24 hr following injury for targeted LC–MS metabolite analysis (Figure