Fig. 3. In vivo effects of PGE₂ on neutrophils during an inflammatory response through genetic manipulation and PGE₂ supplementation.

(A) Morpholino knockdown of endogenous ptges shows the necessity of PGE₂ during inflammation resolution, with a significant increase in neutrophil numbers at the wound site at 8 and 24 hpi. *P < 0.01, two-way analysis of variance (ANOVA) with Bonferonni’s posttest. Data are n = minimum 30 from three experimental repeats. (B) Total neutrophil numbers in unstimulated larvae are reduced (P < 0.05, n = 24 larvae) in the absence of ptges, indicating that the increase during an inflammatory response is not due to increased neutrophils numbers overall. (C) Recapitulation of morphant phenotype in crispant. Three days post fertilization larvae with CRISPR/Cas9-mediated knockdown of ptges display significantly more neutrophils at the wound site at 24 hpi compared to a control-injected guide group targeting tyr. Injured crispants phenocopy ptges morphants. **P < 0.01, ****P < 0.0001, one-way ANOVA with Bonferroni posttest. Data are plotted as means ± SEM with a combined minimum of 40 larvae per group from three experimental repeats. (D) Representative images of crispant larvae at 24 hpi. Area to the right of the magenta dashed line indicates the wound site where GFP neutrophils are counted. Brightfield images demonstrate successful tyr knockdown as a reduction in pigmentation is visible. (E) Dose-response showing increasing concentrations of PGE₂ significantly drive neutrophilic inflammation resolution. PGE₂ was added at 8 hpi with neutrophil counts performed at 12 hpi. Neutrophil numbers at the site of injury are significantly reduced between 0.01 and 1 μM. (F) In the absence of macrophages, exogenous PGE₂ is able to significantly reduce neutrophil numbers back to basal levels at 24hpi. ***P < 0.001, one-way ANOVA with Bonferroni posttest. Minimum of 32 larvae from three repeats. MTZ, metronidazole.