lta4h Deficiency Induces an Immunoregulatory Phenotype Recapitulated by Exogenous LXA4
(A and B) tnf mRNA expression in control and lta4h morphant fish infected with Mm, injected with LTB4, or both. Each bar represents a mean of three independent pools of 20–30 animals. Error bars represent the SD.
(A) tnf mRNA levels in control and morphant fish injected with 110 bacteria and at 1 dpi mock-injected or injected with 1.5 x 10-14 mol LTB4 into the caudal vein 2.5 hr prior to RNA extraction. *p < 0.05; **p < 0.01 (one-way ANOVA. All other comparisons not significant).
(B) tnf mRNA levels in uninfected three dpf control or lta4h morphant animals 2.5 hr after injection of 1.5 x 10-14 mol LTB4 into the caudal vein.
(C) Diagram of LTB4 and LXA4 biosynthetic pathways. Reduction of LTA4H activity (red) is hypothesized to result in increased synthesis of LXA4 through pathway marked by green arrow.
(D) iNOS antibody staining in infected WT and zm5961 animals. Green represents GFP-expressing Mm and red represents iNOS staining. Animals were infected with approximately 100-150 bacteria and fixed for antibody staining and imaging at 3 dpi. The scale bar represents 25 μm.
(E) Numbers of iNOS positive cells in WT and zm5961 animals as described in (D) for both total number of iNOS positive cells in the tail (p = 0.008) and iNOS positive cells within granulomas (p = 0.01).
(F) Light microscopy images of Sudan black stained-neutrophils in right ear of three dpf animals injected with vehicle (left and middle) or 3.5 x 10-14 mol of LXA4 (right) 10 hr prior and with vehicle (left) or 1.5 x 10-14 mol LTB4 (middle and right) into the right ear 4 hr prior. The scale bar represents 50 μm.
(G) Mean number of neutrophils in ears of animals in (F). *p < 0.05; ***p < 0.001 (Kruskal-Wallis nonparametric one-way ANOVA with Dunn′s post-test; other comparisons not significant).
(H) Mean number of neutrophils in ears of three dpf controls, lta4h morphant and zm5961 mutant animals either infected (135 CFU) or mock-infected 1 day prior then injected into right ear with 1.5 x 10-14 mol LTB4 and scored 4 hr later. *p < 0.05; **p < 0.01; ***p < 0.001 (One-way ANOVA with Tukey′s post-test; all other pairwise comparisons not significant).
(I) Mean number of neutrophils recruited to ears of three dpf uninfected animals injected into right ear with 1.5 x 10-14 mol LTB4 after overnight exposure to vehicle or 1 μM PD146176. **p < 0.01; ***p < 0.001 (One-way ANOVA with Tukey′s post-test).
(J) Mean tnf levels relative to uninfected controls in animals (25–30 per group) 8 hr after single dose of 3.5 x 10-14 mol LXA4 or vehicle injected into caudal vein 3 dpi with either mock-infection or with 158 bacteria. Error bars, SD. Representative of three independent experiments.
(K) Mean bacterial burdens (FPC) in 5 dpi WT fish infected at two dpf with 212 CFU and, beginning 1 dpi, given injections of 3.5 x 10-14 mol of LXA4 or vehicle every 12 hr into the caudal vein for 4 days (n = 18 per group). p = 0.0283 (Unpaired t test with Welch′s correction to account for unequal variances).
(L) Examples from (K) of non-cording bacteria within granuloma in vehicle-injected fish and cording bacteria in LXA4-injected fish at 5 dpi. The scale bar represents 30 μm.
(M) Quantitation of cording from (K and L) at 5 dpi. p = 0.018 by Fisher′s exact test of a contingency table.
See also Figure S4.