Figure 5
Figure 5. Dominant-active hif-1αb delays resolution of neutrophilic inflammation. (A) Partial protein alignment showing the conserved hydroxylation sites of Hif-1α. Dominant-active hif-1α has the following nonhydroxylatable mutations: Hif-1αb P402A, P564G, N804A and Hif-1αa P493G, N678A (Hif-1αa lacks the first proline hydroxylation residue). (B-F) Dominant-active forms of hif-1α RNA (177 pg) were injected into the 1-cell-stage zebrafish mpx:GFP embryos, tailfin transection was performed at 2 dpf, and neutrophils counted at 24 hpi. (B) Dominant-active hif-1α caused a significant increase in neutrophil number in the absence of DMOG treatment compared with phenol red–injected negative controls. Dominant-active hif-1αb alone was able to recapitulate the DMOG phenotype, whereas dominant-active hif-1αa homolog did not. 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-active hif-1α variants led to a significant increase in total neutrophil numbers at 2 dpf. Data shown are mean ± SEM, n = 36 performed as 3 independent experiments. (D) Injection of dominant-active hif-1α variants did not alter the recruitment of neutrophils to the injury site after 6 hpi when the tail was transected at 2 dpf. 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. (E) Injection of dominant-active hif-1α led to a significant decrease in percentage of neutrophils at the injury site colabeled with TUNEL apoptosis staining 12 hpi when injured at 2 dpf. Data shown are mean ± SEM, n = 4 performed as independent experiments containing 10 to 25 embryos/injection group/repeat. (F) Number of red (photoconverted) lysosyme C–labeled cells leaving the area of transection at 6 hpi over a time period of 3.5 hpc in phenol red– and dominant-active hif-1α–injected embryos at 2 dpf. 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. (G) Injection of hif-1αb with each hydroxylation site mutated individually shows that the asparagine hydroxylation site of Fih is not required for resolution of neutrophilic inflammation. Mutation of either proline hydroxylation site of Hif-1αb was sufficient to lead to the decrease in neutrophil inflammation resolution. Data shown are mean ± SEM, n = 24 performed as 2 independent experiments. (H) Tg(lyz:Gal4)i252;Tg(UAS:da-hif-1αb-IRES-GFP)i218 positive embryos and wild-type siblings underwent tailfin transection at 3 dpf. Data shown are 24 hpi TSA-positive neutrophil counts, mean ± SEM, n = 30 performed as 3 independent experiments. (I) Tg(lyz:Gal4)i252;Tg(UAS:da-hif-1αb-IRES-GFP)i218 positive embryos had a lower rate of neutrophil apoptosis analyzed by TUNEL staining at 12 hpi after tail transection at 3 dpf. Data shown are mean ± SEM, n = 3 performed as independent experiments containing 9 to 40 embryos/treatment group/repeat.

Dominant-active hif-1αb delays resolution of neutrophilic inflammation. (A) Partial protein alignment showing the conserved hydroxylation sites of Hif-1α. Dominant-active hif-1α has the following nonhydroxylatable mutations: Hif-1αb P402A, P564G, N804A and Hif-1αa P493G, N678A (Hif-1αa lacks the first proline hydroxylation residue). (B-F) Dominant-active forms of hif-1α RNA (177 pg) were injected into the 1-cell-stage zebrafish mpx:GFP embryos, tailfin transection was performed at 2 dpf, and neutrophils counted at 24 hpi. (B) Dominant-active hif-1α caused a significant increase in neutrophil number in the absence of DMOG treatment compared with phenol red–injected negative controls. Dominant-active hif-1αb alone was able to recapitulate the DMOG phenotype, whereas dominant-active hif-1αa homolog did not. 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-active hif-1α variants led to a significant increase in total neutrophil numbers at 2 dpf. Data shown are mean ± SEM, n = 36 performed as 3 independent experiments. (D) Injection of dominant-active hif-1α variants did not alter the recruitment of neutrophils to the injury site after 6 hpi when the tail was transected at 2 dpf. 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. (E) Injection of dominant-active hif-1α led to a significant decrease in percentage of neutrophils at the injury site colabeled with TUNEL apoptosis staining 12 hpi when injured at 2 dpf. Data shown are mean ± SEM, n = 4 performed as independent experiments containing 10 to 25 embryos/injection group/repeat. (F) Number of red (photoconverted) lysosyme C–labeled cells leaving the area of transection at 6 hpi over a time period of 3.5 hpc in phenol red– and dominant-active hif-1α–injected embryos at 2 dpf. 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. (G) Injection of hif-1αb with each hydroxylation site mutated individually shows that the asparagine hydroxylation site of Fih is not required for resolution of neutrophilic inflammation. Mutation of either proline hydroxylation site of Hif-1αb was sufficient to lead to the decrease in neutrophil inflammation resolution. Data shown are mean ± SEM, n = 24 performed as 2 independent experiments. (H) Tg(lyz:Gal4)i252;Tg(UAS:da-hif-1αb-IRES-GFP)i218 positive embryos and wild-type siblings underwent tailfin transection at 3 dpf. Data shown are 24 hpi TSA-positive neutrophil counts, mean ± SEM, n = 30 performed as 3 independent experiments. (I) Tg(lyz:Gal4)i252;Tg(UAS:da-hif-1αb-IRES-GFP)i218 positive embryos had a lower rate of neutrophil apoptosis analyzed by TUNEL staining at 12 hpi after tail transection at 3 dpf. Data shown are mean ± SEM, n = 3 performed as independent experiments containing 9 to 40 embryos/treatment group/repeat.

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