Extremes of LTA4H Expression Drive Hypersusceptibility in Zebrafish (A) Median number of macrophages recruited to the hindbrain ventricle of wild-type and LTA4H-high siblings 4 hr after injection of 150-200 M. marinum into this space at 30 hpf. p = 0.01 (Student′s unpaired t test). Representative of two independent experiments. (B) TNF mRNA levels (mean ± SEM of three independent experiments) for control and LTA4H-high siblings 1 dpi with 150-200 M. marinum. p = 0.02 by Student′s unpaired t test. (C) Fluorescence images of representative wild-type, LTA4H-low and LTA4H-high larvae 3 dpi with 90-100 M. marinum. (D) Bacterial burden of all larvae from (C) by fluorescence pixel counts (FPC). ^**p < 0.01; ^***p < 0.001. (one-way ANOVA with Tukey′s post hoc test; all other comparisons not significant). Representative of > 7 independent experiments measuring differences in bacterial burden of the three genotypes. (E) Mean (±SEM) number of bacteria per infected macrophage in 11 wild-type larvae, 8 LTA4H-low larvae and 13 LTA4H-high larvae, 40 hpi with 150-200 erp mutant M. marinum. ^***p < 0.001 (one way ANOVA with Dunnett′s post hoc test). Representative of two independent experiments. (F) Fluorescence images showing discrete bacterial clumps indicative of macrophage residence in wild-type versus corded extracellular bacteria in their LTA4H-low and LTA4H-high siblings at 3 dpi with 150-200 M. marinum. (G) Percentage of animals in (F) with cording among wild-type, LTA4H-low and LTA4H-high siblings 4 dpi after infection with 90-100 M. marinum. ^*p < 0.05; ^***p < 0.001 (Fisher′s exact test comparing LTA4H-low and LTA4H-high to wild-type). (H) Quantitation of neutral red positive cells 4 dpi after infection with approximately 100 M. marinum in sibling controls and LTA4H-high animals and (I) sibling controls and LTA4H-low animals. See also Figure S2 and Movie S1, Movie S2, and Movie S3.

Modulation of TNF Levels Results in Genotype-Specific Rescue of LTA4H-Mediated Hypersusceptibility

(A) Mean (±SEM) number of bacteria per infected macrophage 40-44 hpi with 150-200 erp mutant M. marinum of wild-type or TNF morphant (MO) siblings.

(B) FPC in control or tnf morphant siblings 3 dpi with 90-100 M. marinum.

(C) Quantitation of frequency of bacterial cording of the animals in (B).

(D) Quantitation of neutral red positive cells 4 dpi after infection with 100 M. marinum in sibling controls or tnf morphants.

(E) Mean (±SEM) number of bacteria per infected macrophage at 40-44 hpi in wild-type animals with or without injection of 0.5 ng rTNF 12 hr after infection with 150-200 erp mutant M. marinum.

(F) FPC in control or rTNF injected siblings 3 dpi with 90-100 M. marinum.

(G) Quantitation of frequency of bacterial cording of the animals in (F).

(H) Quantitation of neutral red positive cells at 4 dpi after infection with 90-100 M. marinum in sibling controls or rTNF injected animals.

(I) FPC in wild-type or lta4h morphant siblings 3 dpi with 90-100 M. marinum per animal after injection of 0.5 ng rTNF at 12 hpi.

(J) Bacterial cording frequency of the animals in (I).

(K) FPC in wild-type animals, LTA4H-high siblings, and LTA4H-high plus TNF morphant animals, at 3 dpi with 90-100 M. marinum.

(L) Quantitation of frequency of bacterial cording in the animals in (K).

Statistical comparisons in panels (I) and (K) by one-way ANOVA with Tukey′s post hoc test; in panels (C),(G),(J) and (L) by Fisher′s exact test; (A),(B),(D),(E),(F),(H) by Student′s unpaired t test. For all panels p < 0.05; p < 0.01; p < 0.001, all other comparisons not significant. See also Figure S3.