Isopimpinellin accelerates inflammation resolution in vivo by inducing neutrophil apoptosis. (A) Inflammation resolution assay in mpx:GFP larvae treated with varying doses of isopimpinellin at 6hpi. Isopimpinellin significantly reduces neutrophil numbers at the wound at 12hpi in a dose-dependent manner (one-way ANOVA with Dunnett′s multiple-comparison post-test; **P<0.01, ***P<0.001; n=18, performed as three independent experiments). Dotted line at y=18.5 indicates mean neutrophil number at wound in DMSO control larvae. (B) Total neutrophil number measured in mpx:GFP larvae treated with DMSO or 25µM isopimpinellin for 24h. Isopimpinellin did not affect total neutrophil number (unpaired t-test; P=0.8696; n=18, performed as three independent experiments). (C) Reverse-migration assay in mpx/Kaede larvae treated with DMSO or 25µM isopimpinellin from 4hpi. Neutrophils at the site of injury were photoconverted at 6hpi and the numbers of photoconverted cells that moved away from the wound were quantified over 5h. Neutrophils migrated away from the wound at a slower rate in isopimpinellin-treated larvae compared to DMSO control larvae. (D) Representative image of isopimpinellin-treated mpx/Kaede larvae at 8hpi (scale bar: 70µm). Solid white line in the left panel indicates the outline of the tail-fin, and the boxed area is magnified in the right-hand panel. White arrows in magnified view indicate neutrophils that appear apoptotic. (E,F) FRET assay in Tg(mpx:FRET)sh237 larvae treated with DMSO or 25µM isopimpinellin from 4hpi and imaged from 6hpi. Cleavage of the caspase-3 target site results in separation of the fluorophores and loss of the FRET signal (red, F). Acceptor (neutrophil) fluorescence (green, F) persists for a further 10-20min before cell death and loss of fluorophore integrity. Time is shown as hours:minutes. Scale bar: 50µm. Number of observable apoptotic events was increased in isopimpinellin larvae (unpaired t-test; ***P<0.001; n=18, performed as three independent experiments). (G) TUNEL assay in mpx:GFP larvae treated with DMSO or 25µM isopimpinellin from 6hpi and fixed at 12hpi. Numbers of TSA-positive neutrophils and TSA/TUNEL double-positive apoptotic neutrophils at the site of injury were measured to calculate percentage neutrophil apoptosis, which was increased in isopimpinellin-treated larvae (unpaired t-test; ***P<0.001; n=115, performed as two independent experiments). (H) Larvae were treated with DMSO, 100µM Z-VAD-FMK (zVAD), 25µM isopimpinellin (Iso) or in combination (Iso+zVAD) from 4hpi and imaged from 6hpi. Number of observable apoptotic events was increased with isopimpinellin alone but the effect was lost with the addition of Z-VAD-FMK (one-way ANOVA with Bonferroni′s multiple-comparison post-test to compare selected columns; *P<0.05; ns, non-significant; n=14, performed as three independent experiments). (I) Larvae were treated with DMSO, 20µM roscovitine or 50µM pyocyanin from 4hpi and imaged from 6hpi. Number of observable apoptotic events was increased with pyocyanin (one-way ANOVA with Bonferroni′s multiple-comparison post-test to compare selected columns; *P<0.05; n=18, performed as three independent experiments).
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