Macrophages are important for host defense and form aggregates in response to S. iniae infection.

(A) Survival of Tg(mpeg1:dendra2) embryos injected at the single-cell stage with either the Irf8 (Irf8 MO) or standard control MO (Ctrl MO) following mock-infection with PBS or infection with 50 CFU S. iniae. Irf8 morphants infected with both WT and cpsA S. iniae have impaired survival (p < 0.0001), compared to control morphants. (B) S. iniae labelled with Cell Tracker Red are found within macrophage aggregates. Scale bar is 20μm. (C) S. iniae infection results in the development of macrophage aggregates in the trunk/tail of a proportion of infected fish by 24 hpi (diagram, red box). Representative 20X images of macrophage aggregates in Tg(mpeg1:dendra2) larvae 24 hpi following infection with PBS, cpsA mutant, 10 CFU, 50 CFU, and 100 CFU WT S. iniae as indicated. Scale bar is 80 μm. (D) Quantification of the average total percent of larvae forming macrophage aggregates from (C). (E) Average number of aggregates per larvae and (F) average aggregate size, as measured by the peripheral area of aggregates, at 24 hpi following infection with 50 or 100 CFU WT S. iniae. Area and number were not statistically significant across conditions. Data are from at least 3 independent experiments, with 24 larvae per condition.

S. iniae capsule is a determinant of aggregate formation.

(A) Representative 20X images of larvae at 24 hpi following inoculation with either PBS, 50 CFU WT S. iniae alone; 50 CFU equivalent heat killed (HK) or formalin killed (FK) plus 100 CFU cpsA S. iniae; 100 CFU cpsA alone; or 100 CFU live cpsA plus 100 CFU equivalent of HK cpsA. Scale bar is 80 μm. (B) Quantification of the average total percent of larvae forming macrophage aggregates from (A).

Disruption of Lta4h signaling abrogates macrophage aggregation.

(A) NFκB reporter expression in Tg(mpeg1:mCherry) larvae at 24hpi following inoculation with PBS or 50 CFU S. iniae. Aggregate macrophages and adjacent cells show NFκB expression. Images at 40X; scale bar is 20 μm. Non-specific signal is indicated by yellow asterisks. (B and C) Average percentage of total larvae with macrophage aggregates at 24 hpi following inoculation with 50 CFU WT S. iniae. Aggregates fail to form in (B) Lta4h knockdown, p = 0.0087, average of 6 independent experiments; during (C) Lta4h inhibition with 100 μM Bestatin, p = 0.0022, average of 6 independent experiments; as opposed to relevant controls (Control MO, 0.1% DMSO, respectively), and were decreased in an (D) Lta4h mutant, p = 0.1000, average of 3 independent experiments. (E) RT-PCR of lta4h from mRNA extracted from 2–4 dpf zebrafish. The arrow denotes the presence of an alternative transcript in larvae injected with a splice-blocking Lta4h morpholino. Lane 1 = Ctrl MO, 2 = 100 μM Lta4h MO, 3 = 200 μM Lta4h MO, 4 = 500 μM Lta4h MO. (F) Representative 20X images of exogenous addition of LTB4 rescuing aggregate formation in Lta4h morphants. Larvae were treated with 30 nM LTB4 or 0.1% ethanol (EtOH). Scale bar is 80 μm. (G) Quantification of the average total percent of larvae forming macrophage aggregates from (F).

Neutrophil crosstalk drives macrophage aggregate formation.

(A) Double transgenic Tg(mpeg:dendra2) x Tg(mpx:mCherry) larvae show that neutrophils are present in and around macrophage aggregates. Images at 40X; scale bar is 20 μm. (B) Representative 20X images of Rac2WT or Rac2D57N larvae at 24 hpi following inoculation. Tg(mpx:mCherry-2a-rac2wt) (Rac2WT) or Tg(mpx:mCherry-2a-rac2d57n) (Rac2D57N) were crossed to Tg(mpeg1:dendra2) and the resulting double transgenic larvae were infected with 50 CFU WT S. iniae or mock-infected with PBS. Rac2D57N larvae are defective for aggregate formation. Scale bar is 80 μm. (C) Quantification of the average total percent of larvae forming macrophage aggregates from (B).

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.

(A) Representative 63X images of macrophage aggregates in double transgenic Tg(mpeg:dendra2) x Tg(lyz:lta4h-2a-mCherry) larvae. Scale bar is 20 μm. (B) Average aggregate size, as measured by the peripheral area of aggregates, and (C) average number of aggregates per larvae at 24 hpi following infection with 50 CFU S. iniae in control or Lta4h morphants in a WT or Tg(lyz:lta4h) (lyz:lta4h) larvae background. Area and number were not statistically significant across all conditions.