Sterile injury-induced neutrophilic inflammatory responses are exacerbated in the absence of CFTR. (A–C) Neutrophil chemotaxis to sterile tissue lesions in WT, cftr –/– mutant (cftr –/–) and cftr morphant (cftr MO) TgBAC(mpx:EGFP)i114 zebrafish larvae. (A) Larvae were tail amputated then the number of neutrophils mobilized to the site of injury (dotted lines) has been observed and counted by fluorescent microscopy throughout inflammation. The wound area is defined as the region between the amputation edge and caudal hematopoietic tissue end (CHT). (B) Dynamics of neutrophil recruitment towards the wound over 24 h (n = 30; two-way ANOVA with Tukey post-test). (C) Representative confocal images of injured tails at 4 hpi (scale bars, 200 μm). (D,E) Total number of neutrophils (D) in whole TgBAC(mpx:EGFP)i114 larvae (n = 18; one-way ANOVA with Dunnett's post-test). (E) Microscopy revealed a disorganized neutrophilic distribution in CFTR-depleted animal compared to control counterpart (scale bars, 200 μm). (F) Neutrophil mobilization into the otic cavity (oc, dotted lines) in response to DMSO or fMLP injection in TgBAC(mpx:EGFP)i114 larvae monitored at 2 h-post injection. Representative photomicrographs of fMLP-injected animal compared to DMSO-injected control (top panel; scale bars, 50 μm). Neutrophil counts (n = 18; one-way ANOVA with Dunnett's post-test) (bottom panel). See also Supplementary Figure 1.

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.

CFTR deficiency delays inflammation resolution in vivo both by reducing neutrophil apoptosis and reverse migration of neutrophils. (A,B)Control and cftr MO larvae were amputated and stained with TUNEL/TSA to label apoptotic cells. (A) Representative confocal pictures of injured tails at 8 hpi (scale bars, 60 μm) revealing the proportion of apoptotic neutrophils at the wound (white arrow). (B) Quantification of neutrophil apoptosis rate at site of injury at 4 and 8 hpi. (n = 18–22, Fisher t-test). (C–F) Neutrophil reverse migration in control and cftr MO larvae. (C) Tail transection was performed on 3 dpf Tg(mpx:gal4)sh267;Tg(UASkaede)i222 larvae. The site of injury was photoconverted at 4 hpi, then the number of photoconverted neutrophils (red) moving away (white dotted box) from the wound (blue dotted box) were time-lapse imaged and quantified over 4 h post-photoconversion by confocal microscopy. (D) Representative confocal imaging of amputated tails showing the kinetics of photoconverted neutrophils that migrate away from photoconverted region over inflammation resolution. (E) Plot showing the number of photoconverted neutrophils leaving the area of injury over 4 h post photoconversion in control and cftr MO. Line of best fit shown is calculated by linear regression. P-value shown is for the difference between the 2 slopes (n = 12, performed as 3 independent experiments). (F) Representative confocal imaging of amputated tails showing the kinetics of new neutrophils (green) recruited towards site of injury after photoconversion. See also Supplementary Figure 3.

Neutrophilic response in CF hampers tissue repair in vivo. (A,B) Tail fin regeneration assessment in WT, cftr –/– and cftr MO zebrafish. Two dpf embryos were tail fin amputated then the potential of tail fin regeneration is evaluated by measuring regenerated fin areas at 3 dpi (blue). (A) Measurement of regenerated fin areas (n = 30, one-way ANOVA with Dunnett's post-test). (B) Representative imaging of injured tail fin at 3 dpi (scale bars, 200 μm). (C) Selective ablation of neutrophils has been carried out in WT and CF animals by injecting csf3r-MO. Measurement of regenerated fin areas in the presence or absence of neutrophils (n = 21, one-way ANOVA with Tukey post-test). (D)TgBAC(mpx:EGFP)i114 larvae were pretreated with DPI or H₂O₂ prior to tail fin amputation procedures, then injured and immediately put back in treatments for 4 h. Regenerated fin areas are measured at 3 dpi (n = 21, one-way ANOVA with Dunnett's post-test). (E) Inhibition of NADPH oxidase activity by injecting duox2-MO has been carried out in WT and CF animals. Embryos were tail fin injured and regenerated fin areas measured at 3 dpi (n = 21, two-tailed Bonferroni t-test).

TIIA-driven neutrophil apoptosis and reverse migration accelerate inflammation resolution in CF. (A)TgBAC(mpx:EGFP)i114 larvae were pretreated with 25 μM of TIIA prior to tail fin amputation procedure, then injured and immediately put back in treatments for 4 h. Neutrophil number at the wound was counted at 8 hpi (n = 21, two-tailed Bonferroni t-test). (B,C)TgBAC(mpx:EGFP)i114 larvae were injured and treated from 4 hpi with 25 μM of TIIA. (B) Neutrophil number at wound was counted at 8 hpi (n = 21, two-tailed Bonferroni t-test). (C) representative number of neutrophils remaining at wounds at 8 hpi (scale bars, 200 μm). (D) Neutrophil apoptosis quantification at 8 hpi in cftr MO treated with 25 μM of TIIA from 4 hpi and stained with TUNEL/TSA. (n = 40, Fisher t-test). (E) Reverse-migration assay in cftr MO Tg(mpx:gal4)sh267;Tg(UASkaede)i222. At 4 hpi fish were treated with 25 μM of TIIA and neutrophils at site of injury were photoconverted. The numbers of photoconverted cells that moved away from the wound were time-lapse imaged and quantified over 4 h. (F,G) Regenerative performance after TIIA treatment. (F) Regenerated fin areas are measured at 3 dpi (n = 21, two-tailed Bonferroni t-test). (G) Representative imaging of injured tail fin at 3 dpi (scale bars, 200 μm). (H) Schematic diagram showing TIIA efficiently accelerates inflammation resolution by inducing neutrophil apoptosis and reverse migration at wounds and improves tissue repair in CF animal.

Proposed model showing CF-related inflammation immuno-pathogenesis. The spatiotemporal events associated with CFTR ablation reveal a mechanism whereby CFTR participates in the adjustment of innate immunity, conditioning key regulatory mediators involved in the regulation of inflammation and tissue repair. A sterile lesion is characterized by early release of epithelial H₂O₂ through DUOX2, leading to neutrophil recruitment towards wounds; coordination of neutrophil apoptosis and reverse migration promoting efficient inflammation resolution, and allowing to restore tissue homeostasis and initiate tissue repair. In contrast, in CF zebrafish sterile injury leads to extensive inflammation, typified by increased then sustained accumulation of neutrophils at wounds: (1) excessive epithelial ROS release drives increased neutrophil recruitment towards wounds; (2) reduction of neutrophil apoptosis and impaired retrograde migration of neutrophils resulting in delayed resolution of inflammation. (3) Therefore, the increased number of neutrophils that mobilize in an uncontrolled manner at wound sites causes persistent inflammation, severe tissue damage, and abnormal tissue repair.