Neutrophil-specific expression of Lta4h rescues macrophage aggregation and correlates with host survival in Lta4h-deficient larvae.

<p>(A) Schematic of the <i>Tol2-lyz</i>:<i>lta4h-2a-mCherry</i> construct that was injected into AB-WT embryos to generate the <i>Tg(lyz</i>:<i>lta4h-2a-mCherry)</i> transgenic line. (B) The transgenic Lta4h sequence is non-targetable by morpholino knockdown. <i>Tg(lyz</i>:<i>lta4h-2a-mCherry)</i> (lyz:lta4h) or <i>Tg(mpx</i>:<i>mCherry)</i> (WT) was crossed to <i>Tg(mpeg1</i>:<i>dendra2)</i> and injected with either the Ctrl MO or Lta4h MO and monitored for macrophage aggregation at 24 hpi. Representative 20X images of WT or <i>lyz</i>:lta4h larvae at 24 hpi following inoculation with 50 CFU WT <i>S</i>. <i>iniae</i>, 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 <i>lyz</i>:<i>lta4h</i> larvae in either control or Lta4h morphant larvae infected with 50 CFU <i>S</i>. <i>iniae</i>. Compared with WT Ctrl MO larvae (black solid line), WT Lta4h MO larvae (black dotted line) have worse survival (p = 0.00437). Compared with <i>lyz</i>:<i>lta4h</i> Ctrl MO larvae (magenta solid line), <i>lyz</i>:<i>lta4h</i> Lta4h MO larvae (magenta dotted line) do not have significantly worse survival. However, <i>lyz</i>:<i>lta4h</i> 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 <i>lyz</i>:<i>lta4h</i> is macrophage dependent. Survival of WT or <i>lyz</i>:<i>lta4h</i> larvae in either control or Irf8 morphant larvae (lacking macrophages) infected with 50 CFU <i>S</i>. <i>iniae</i>. Compared with <i>lyz</i>:<i>lta4h</i> 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), <i>lyz</i>:<i>lta4h</i> 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.