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

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 TNF alpha+ macrophages had lower NAD(P)H mean lifetime and were more oxidized compared to TNF alpha- 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 TNF alpha+ wound macrophages, made intracellular redox state more reduced and improved tissue repair. By contrast, depletion of STAT6 increased TNF alpha+ 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.

Automated summary

This study used two-photon imaging of autofluorescent metabolic coenzymes NAD(P)H and FAD to assess macrophage metabolism in the wound microenvironment. Results showed that inhibiting glycolysis led to a more oxidized intracellular redox state in macrophages, and infection and thermal injury induced a more oxidized redox state in wounded tissues. Kinetic analysis revealed a shift toward a more reduced redox state during tissue repair. Metformin improved tissue repair by reducing TNF alpha+ wound macrophages and making the intracellular redox state more reduced, while depletion of STAT6 impaired regeneration by increasing TNF alpha+ wound macrophages and making the redox state more oxidized. The study suggests that autofluorescence of NAD(P)H and FAD can be used to probe the temporal and spatial regulation of macrophage metabolism during tissue damage and repair.