Fig. 6

Fig. 6

NADPH oxidase inhibitor Apocynin increases susceptibility to notochord infection.

(A-B) E. coli-GFP were injected in the notochord of 2 dpf tg(mpx:GFP) embryos in DMSO or Apocynin treatment conditions. (A) At 1 dpi, superoxide production was visualised using DHE (red), neutrophils and E. coli were detected using GFP. Notochord images are representative maximum projection of fluorescence confocal images overlaid with transmitted light images. Pink arrowheads show DHE⁺ neutrophils, white arrows show DHE^- neutrophils and white arrowheads: E. coli, scale bars: 30 μm. (B) Quantification of DHE-positive cells in DMSO and Apocynin treated larvae (mean ±SEM, N_larvae = 5 per condition, Mann-Whitney test, one-tailed, * p<0.05). (C, D) Tg(mpx:GFP) embryos were treated with Apocynin (APO) or DMSO at 2 dpf. Neutrophils (GFP) were imaged using fluorescent microscopy at 6 hours post-treatment (hpT) and 24 hpT. (C) Representative fluorescent images of Apocynin or DMSO treated larvae at 24 hpT. Scale bar: 400 μm. (D) Corresponding counts of total neutrophil population in indicated conditions (mean ± SEM, N_DMSO = 31 and N_APO = 29, Mann-Whitney test, two-tailed, * p<0.05, representative of 2 independent experiments). (E, F, G) Two dpf tg(lyz:DsRed) embryos were infected in the notochord with E.coli-GFP and treated with Apocynin. (E) Neutrophils (DsRed) and E. coli (GFP) were imaged repeatedly in individual larvae using fluorescent microscopy at 6 hpi and 1 dpi. Bacteria (white arrowheads) were present at 6 hpi in both DMSO- and Apocynin-treated embryos. At 1 dpi, bacteria disappeared in DMSO-treated embryos (arrows) while their number increased in Apocynin-treated embryos (white arrowheads). (F) Infection outcome of E. coli infected embryos after in DMSO or Apocynin treatments were scored from 0 to 3 dpi (the number of larvae is indicated in the columns). (G) Survival curves of larvae uninfected and infected with E. coli from 0 to 3 dpi in DMSO or Apocynin treatments. (N_larvae is indicated in the figure, log rank test, p<0.001, from two independent experiments). (H) Two dpf mpx^+/+ or mpx^-/- embryos were infected in the notochord with E. coli-GFP and treated either with DMSO or Apocynin (APO). Infection outcome of E. coli infected embryos were scored from 0 to 2 dpi (the absolute number of larvae is indicated in the columns). (I) Survival curves of mpx^+/+ or mpx^-/- larvae infected with E. coli from 0 to 2 dpi in DMSO or Apocynin treatments (N_larvae is indicated in the figure, log rank test, p<0.01, from two independent experiments).