Figure 2

Exuberant wound-induced oxidative responses drive the overactive neutrophilic response in CF larvae. (A,B) WT, cftr –/– and cftr MO stained with CellROX® to label H₂O₂ production. Means ± SEM ROS intensity at 30 min post-injury (mpi) (A) and associated pseudocolored photomicrographs of uninjured and injured tails revealing ROS production (B) (n = 15; one-way ANOVA with Dunnett's post-test). (C) mRNA levels of duox2 gene in tail fin tissue at 30 mpi. Gene expression was expressed as fold change over tail fin tissue from uninjured larvae (30 fins per replicate; mean relative ± SEM gene expression; two-tailed Student t-test). (D)TgBAC(mpx:EGFP)i114 controls, cftr, duox2, and double cftr/duox2 morphants were tail amputated and neutrophils at wounds were enumerated at 2 and 4 hpi. (n = 21; two-way ANOVA with Tukey post-test). (E) Representative photomicrographs of injured tails at 4 hpi (scale bars, 200 μm). (F) WT, cftr –/– and cftr MO TgBAC(mpx:EGFP)i114 larvae were pretreated with DPI or H₂O₂ prior tail amputation procedure, then injured and immediately put back in treatments. Neutrophil counts at 4 hpi (n = 21; one-way ANOVA with Dunnett's post-test). (G) Schematic diagram showing inhibition of ROS signaling efficiently reduces neutrophil inflammation at wounds in CF animal. (H)TgBAC(mpx:EGFP)i114 larvae were treated with DPI at 4 hpi. Neutrophil counts at 8 hpi (n = 21; two-tailed Bonferroni t-test). See also Supplementary Figure 2.