|
Fig. 2 Injury-induced inflammatory responses are exacerbated in the absence of Nrf2 signaling in zebrafish larvae. (A) Schematic of injury-induced inflammation assay and analyses. Caudal fin amputations (dotted lined) were performed proximally through the notochord (N) at 3 days post-fertilization (dpf). (B) Control (control morphant (MO)) and cftr MO (generated using the validated cftr morpholino [15]) were caudal fin amputated then the level expressions of mRNA for nrf2a (left) and nrf2b (right) genes are evaluated at 2 hours post-amputation (hpA) by RT qPCR. Graphs showing the fold change over total tissue from uninjured larvae (relative gene expression from at least 3 independent experiments performed in triplicates, mean and quartiles shown, Two-tailed Student t-test). (C–E) Neutrophil mobilization to tissue injury in the neutrophil-specific TgBAC(mpx:EGFP)i114 line [18] is observed and enumerated at 2 hpA under a fluorescence microscope. The neutrophil count area is defined as the region between the blood vessels (BV) end and the amputation edge (AE). (C) Number of neutrophils mobilized to the wound in control MO, nrf2a MO, nrf2b MO or double nrf2a MO / nrf2b MO (nrf2a+b MO). Each dot represents the number of neutrophils at the wound in a single larva (3 independent experiments, One-way ANOVA with Dunnett’s comparisons test). (D) Representative images of injured tails (scale bars, 100 μm). The images are denoted as green data points in (C). (E) Inhibition of nrf2a, nrf2b or nrf2a+b was carried out in control and CF animals by injecting the nrf2a morpholino, the nrf2b morpholino or the nrf2a + nrf2b morpholinos reciprocally. Neutrophil recruitment assay (3 independent experiments, Two-way ANOVA with Tukey’s multiple comparisons test). (F) Control MO and cftr MO TgBAC(mpx:EGFP)i114 were treated with the NRF2 inhibitor ML 385, the NRF2 agonist CDDO-Me or DMSO as mock control prior to caudal fin amputation procedure, then injured and immediately put back in treatments. Neutrophil number at injured tails were enumerated at 2 hpA (3 independent experiments, Two-way ANOVA with Tukey's multiple comparisons test). (G) Relative expression of mRNA for duox, nqo1, hmox1, cxcl8 and il1β at 2 hpA (duox, cxcl8 and il1β) or 8 hpA (nqo1 and hmox1) were determined by RT qPCR. (from at least 3 independent experiments performed in triplicates,mean and quartiles shown, Two-tailed Student t-test).