Neutrophil-specific expression of Lta4h rescues macrophage aggregation and correlates with host survival in Lta4h-deficient larvae.
(A) Schematic of the Tol2-lyz:lta4h-2a-mCherry construct that was injected into AB-WT embryos to generate the Tg(lyz:lta4h-2a-mCherry) transgenic line. (B) The transgenic Lta4h sequence is non-targetable by morpholino knockdown. Tg(lyz:lta4h-2a-mCherry) (lyz:lta4h) or Tg(mpx:mCherry) (WT) was crossed to Tg(mpeg1:dendra2) and injected with either the Ctrl MO or Lta4h MO and monitored for macrophage aggregation at 24 hpi. Representative 20X images of WT or lyz:lta4h larvae at 24 hpi following inoculation with 50 CFU WT S. iniae, in both control and Lta4h morphant larvae. Scale bar is 80 μm. (C) Quantification of the average total percent of larvae forming macrophage aggregates from (B). (D) Survival of WT or lyz:lta4h larvae in either control or Lta4h morphant larvae infected with 50 CFU S. iniae. Compared with WT Ctrl MO larvae (black solid line), WT Lta4h MO larvae (black dotted line) have worse survival (p = 0.00437). Compared with lyz:lta4h Ctrl MO larvae (magenta solid line), lyz:lta4h Lta4h MO larvae (magenta dotted line) do not have significantly worse survival. However, lyz:lta4h Lta4h MO larvae (magenta dotted line) have significantly better survival than WT Lta4h MO larvae (black dotted line, p = 0.0142). (E) Increased survival in lyz:lta4h is macrophage dependent. Survival of WT or lyz:lta4h larvae in either control or Irf8 morphant larvae (lacking macrophages) infected with 50 CFU S. iniae. Compared with lyz:lta4h Ctrl MO larvae (green solid line), lyz:lta4h Irf8 MO larvae (green dotted line) have significantly worse survival (p < 0.0001). Compared with WT Irf8 MO larvae (black dotted line), lyz:lta4h Irf8 MO larvae do not have a significant difference in survival. Data are statistically pooled from at least 3 independent experiments, each with 24 larvae per condition.