Figure 4
Figure 4. DMOG-induced delay in inflammation resolution is blocked by genetic inhibition of the Hif-1α pathway. (A) The 24 hpi neutrophil counts in the mpx:GFP line at 2 dpf after injection with control and arnt-1 morpholinos. DMSO and DMOG treatment was performed at 4 hpi. Data shown are mean ± SEM, n = 12 performed as 2 independent experiments. (B-E) We injected 177 pg of dominant-negative hif-1α RNA into the 1-cell-stage zebrafish mpx:GFP embryos, and then neutrophil counts were performed at 24 hpi after tail transection at 2 dpf. (B) Injection of dominant-negative hif-1αb abrogated the increase in neutrophil number at the site of injury at 24 hpi seen with DMOG treatment, whereas embryos injected with phenol red as a negative control, or dominant-negative hif-1αa alone exhibited a significant increase in neutrophil number after DMOG treatment. Data shown are mean ± SEM, n = 24 performed as 2 independent experiments. P values were calculated using 1-way ANOVA and Bonferroni multiple comparison test, where P < .05, **P < .01, and ***P < .001. (C) Injection of dominant-negative hif-1α variants led to no significant difference in total neutrophil number at 2 pf compared with phenol red–injected negative control embryos. Data shown are mean ± SEM, n = 36 performed as 3 independent experiments. (D) Injection of dominant-negative hif-1α caused no significant change in percentage of neutrophils at the injury site colabeling with TUNEL stain (12 hpi injured at 2 dpf). Data shown are mean± SEM, n = 4 performed as independent experiments containing 5 to 25 embryos/injection group/repeat. (E-F) Dominant-negative hif-1α can block the increased neutrophil retention at the site of injury caused by DMOG treatment. Embryos were injected with dominant-negative hif-1αb, grown to 3 dpf, and imaged 3.5 hours after photoconversion, with only the red channel shown, as a binary image. (E, left panels) In DMSO-treated larvae, red-labeled neutrophils have migrated away from the site of transection. (E, right panel) In DMOG-treated larvae, red-labeled neutrophils also move away from the site of transection. (F) The number of red (photoconverted) lysozyme C–labeled cells leaving the area of transection at 6 hpi over a time period of 3.5 hpc in DMSO- and DMOG-treated embryos at 2 dpf that were injected at the 1-cell stage with dominant-negative hif-1αb. Data shown are mean ± SEM, n = 14 performed as 3 independent experiments. Line of best fit shown is calculated by linear regression. P value shown is the difference between the 2 slopes.

DMOG-induced delay in inflammation resolution is blocked by genetic inhibition of the Hif-1α pathway. (A) The 24 hpi neutrophil counts in the mpx:GFP line at 2 dpf after injection with control and arnt-1 morpholinos. DMSO and DMOG treatment was performed at 4 hpi. Data shown are mean ± SEM, n = 12 performed as 2 independent experiments. (B-E) We injected 177 pg of dominant-negative hif-1α RNA into the 1-cell-stage zebrafish mpx:GFP embryos, and then neutrophil counts were performed at 24 hpi after tail transection at 2 dpf. (B) Injection of dominant-negative hif-1αb abrogated the increase in neutrophil number at the site of injury at 24 hpi seen with DMOG treatment, whereas embryos injected with phenol red as a negative control, or dominant-negative hif-1αa alone exhibited a significant increase in neutrophil number after DMOG treatment. Data shown are mean ± SEM, n = 24 performed as 2 independent experiments. P values were calculated using 1-way ANOVA and Bonferroni multiple comparison test, where P < .05, **P < .01, and ***P < .001. (C) Injection of dominant-negative hif-1α variants led to no significant difference in total neutrophil number at 2 pf compared with phenol red–injected negative control embryos. Data shown are mean ± SEM, n = 36 performed as 3 independent experiments. (D) Injection of dominant-negative hif-1α caused no significant change in percentage of neutrophils at the injury site colabeling with TUNEL stain (12 hpi injured at 2 dpf). Data shown are mean± SEM, n = 4 performed as independent experiments containing 5 to 25 embryos/injection group/repeat. (E-F) Dominant-negative hif-1α can block the increased neutrophil retention at the site of injury caused by DMOG treatment. Embryos were injected with dominant-negative hif-1αb, grown to 3 dpf, and imaged 3.5 hours after photoconversion, with only the red channel shown, as a binary image. (E, left panels) In DMSO-treated larvae, red-labeled neutrophils have migrated away from the site of transection. (E, right panel) In DMOG-treated larvae, red-labeled neutrophils also move away from the site of transection. (F) The number of red (photoconverted) lysozyme C–labeled cells leaving the area of transection at 6 hpi over a time period of 3.5 hpc in DMSO- and DMOG-treated embryos at 2 dpf that were injected at the 1-cell stage with dominant-negative hif-1αb. Data shown are mean ± SEM, n = 14 performed as 3 independent experiments. Line of best fit shown is calculated by linear regression. P value shown is the difference between the 2 slopes.

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