Figure 6. Macrophage metabolic switches are associated with changes in tissue repair.

(A–B) Thermal injury distal to the notochord was performed using N-phenylthiourea-treated double transgenic wild-type (wt) or stat6-deficient zebrafish larvae (Tg(tnf:GFP x mpeg1:mCherry-CAAX)) at 3 days post fertilization (dpf); larvae were fixed at 72 and 96 hr post burn (hpb). (A) Representative images of sum projections of z-stacks acquired by spinning disk confocal microscopy are shown; scale bar = 100 μm. (B) TNFα expression in macrophages was quantified by scoring cells for GFP signal and is displayed as proportion of TNFα+ cells per larva with bars showing arithmetic mean and 95% CI; results are from three biological repeats (wt = 33, stat6−/− = 46 at 72 hpb, wt = 33, stat6−/− = 20 larvae at 96 hpb). Larvae were generated by a het incross and genotyped post imaging; only wt and stat6−/− larvae were analyzed. (C–F) Thermal injury distal to the notochord was performed using transgenic zebrafish larvae (Tg(mpeg1:mCherry-CAAX)) in wt- or stat6-deficient background at 3 dpf. Autofluorescence imaging of nicotinamide adenine dinucleotide (phosphate) (NAD(P)H) and flavin adenine dinucleotide (FAD) was performed on live larvae at 72 hpb. (C) Representative images of mCherry expression to show macrophages, optical redox ratio, and NAD(P)H and FAD mean lifetimes (${\displaystyle {\tau }_m}$) are shown; 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 (D) optical redox ratio, (E) Optical Metabolic Imaging index, and (F) NAD(P)H mean lifetime (${\displaystyle {\tau }_m}$) from three biological repeats (wt = 217 cells/12 larvae, stat6−/− = 272 cells/12 larvae) is shown; quantitative analysis of associated NAD(P)H and FAD mean (${\displaystyle {\tau }_m}$) and individual lifetime endpoints (${\displaystyle {\tau }_1,{\tau }_2,{\alpha }_1}$), and sample size for each repeat are included in Figure 6—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 6—source data 1. (G) Control or metformin-treated larvae were fixed at 72 hpb following thermal injury of the tail fin. Representative single-plane brightfield images are shown; scale bar = 100 μm. (H) Quantitative analysis of tail fin tissue regrowth area per larva, displayed with bars showing arithmetic mean and 95% CI. Results are from three biological repeats (control = 59, metformin = 60 larvae). (I) Wt- or stat6-deficient larvae were fixed at 96 hpb following thermal injury of the tail fin. Representative single-plane brightfield images are shown; scale bar = 100 μm. (J) Quantitative analysis of tail fin tissue regrowth area per larva, displayed with bars showing arithmetic mean and 95% CI. Results are from three biological repeats (wt = 36, stat6−/− = 24 larvae). Larvae were generated by a het incross and genotyped post imaging; statistical analysis was performed on wt, stat6± and stat6−/−; only wt and stat6−/− larvae are shown.

Figure 6—figure supplement 1. Individual nicotinamide adenine dinucleotide (phosphate) NAD(P)H and flavin adenine dinucleotide (FAD) fluorescence lifetime endpoints associated with Figure 6.

(A) Total number of macrophages in whole larvae displayed with bars showing arithmetic mean and 95% CI; larvae were generated by a het incross and genotyped post imaging; results are from three biological repeats (wild-type (+/+) = 26, stat6+/− = 52, stat6−/− = 24). (B) Number of macrophages at the burn wound with bars showing arithmetic mean and 95% CI. Quantitative analysis of (C) tau1 (${\displaystyle {\tau }_1}$), free/short lifetime of NAD(P)H, (D) tau2 (${\displaystyle {\tau }_2}$), bound/long lifetime of NAD(P)H, (E) alpha1 (α₁), fractional component of free NAD(P)H, (F) mean lifetime (${\displaystyle {\tau }_m}$) of FAD, (G) tau1 (${\displaystyle {\tau }_1}$), bound/short lifetime of FAD, (H) tau2 (${\displaystyle {\tau }_2}$), free/long lifetime of FAD and (I) alpha1 (α₁), fractional component of bound FAD. p values represent statistical analysis of the overall effects. Estimated means with 95% CI and overall effects with p values are included in Figure 6—source data 1. (J) Sample size of autofluorescence imaging data set shown in Figure 6 and this supplement.