Intestinally-derived Saa regulates systemic neutrophil activity.

(A) qRT-PCR of saa from whole 6 dpf larvae of the indicated genotypes (n = 4 replicates / genotype, 25–30 larvae / replicate) (B) Enumeration of intestine-associated lyz:DsRed+ neutrophils along the anterior to posterior axis (segment 1 to segment 3) in 6 dpf larvae (n ≥ 25 larvae / genotype). (C) lyz:DsRed+ neutrophil recruitment to caudal fin wound 6 hours following amputation in 6 dpf zebrafish larvae (n ≥ 25 larvae / genotype at 6 hour time point). (D) CFU quantification of bacterial concentration following 4 hour co-culture of lyz:DsRed+ adult zebrafish neutrophils with P. aeruginosa (P.a., MOI 0.2) (8 replicates / genotype). (E) Representative stereoscope images of IEC specific mCherry expression in 6 dpf Tg(-0.349cldn15la:mCherry)rdu65 larvae compared to non-transgenic (NTG) controls. White dashed line indicates the intestine (scale bar = 500 μm). (F) Representative confocal micrograph of immunostained transverse section of Tg(-0.349cldn15la:mCherry) 6 dpf larvae labeled with the absorptive cell brush border-specific antibody 4E8 (scale bar = 20 μm). (G,H) qRT-PCR of cldn15la, fabp2, and fabp10a (G) from sorted Tg(-0.349cldn15la:mCherry)+ IECs and saa (H) from cldn15la:mCherry+ and negative cells isolated from 6 dpf larvae of indicated genotypes (13,000 –0.349cldn15la:mCherry+ or mCherry negative cells / replicate, 4 replicates / genotype, 30 larvae / replicate). (I) qRT-PCR of saa from 6 dpf larval dissected digestive tissue of the indicated genotypes (n = 4 replicates / genotype, 25–30 larvae / replicate) (J) Enumeration of intestine-associated lyz:DsRed+ neutrophils in 6 dpf larvae (n = 30 larvae / genotype). (K) lyz:DsRed+ neutrophil recruitment to caudal fin wound 6 hours following amputation in 6 dpf zebrafish larvae (n ≥ 18 larvae / genotype at 6 hour time point). (L) CFU quantification of bacterial concentration following 4 hour co-culture of lyz:DsRed+ adult zebrafish neutrophils with P. aeruginosa (P.a., MOI 0.2) (3–6 replicates / genotype). (M) il1b qRT-PCR from lyz:DsRed+ neutrophils co-cultured with and without P.a. ex vivo for 4 hours (n ≥ 2 replicates / condition). (N) CFU quantification of in vivo P.a. bacterial burden following systemic infection of larval zebrafish at 5 days post infection (dpi) (data from 3 independent experiments, n ≥ 30 larvae / genotype). Data in panels A-D and H-N were analyzed by one-way ANOVA with Tukey’s multiple comparisons test. A Mann-Whitney test was applied to panel G. For panels C and K, statistical comparisons were performed amongst samples within the same time point. Data are presented as mean ± SEM. * p p p p 0.0001.