Fig. 1.

Zebrafish exposure to nonlethal Shigella dose induces generation of more resilient neutrophils.

(A) Training of 2 dpf zebrafish larvae. (B) Representative images of Tg(lyz::dsRed) larvae at 48 hp1i. Scale bar, 500 μm. (C) Quantification of neutrophils [Tg(lyz::dsRed)] in naïve and Shigella-trained (2 × 10³ ± 1.4 × 10³ CFUs) larvae. N = 3 with 10 larvae per time point (mean ± SD). (D) Quantification of macrophages [Tg(mpeg::mCherry)] in naïve and Shigella-injected (1.8 × 10³ ± 8.6 × 10² CFUs) larvae. N = 3 with 11 larvae per time point (mean ± SD). (E) Expression of cxcl8a, cxcl18b, il1b, and il10 in Shigella-trained larvae (1.3 × 10³ ± 5.5 × 10² CFUs) as compared to naïve larvae. N = 3 with >5 larvae per time point (mean ± SEM). (F) Reinfection of trained larvae with lethal dose injection of Shigella at 4 dpf. (G) Representative images of Tg(lyz::dsRed) naïve and Shigella-trained larvae reinfected with Shigella flexneri M90T. Scale bar, 50 μm. (H) Quantification of recruited neutrophils [Tg(lyz::dsRed)] to the HBV in naïve and Shigella-trained larvae following reinfection with Shigella (PBS, 2.6 × 10⁴ ± 9 × 10³ CFUs; Shigella, 2.7 × 10⁴ ± 1 × 10⁴ CFUs). N = 3 with 5 larvae per time point (mean ± SD). (I) Bacterial counts from naïve and Shigella-trained larvae reinfected with Shigella (PBS, 2.7 × 10⁴ ± 4.5 × 10³ CFUs; Shigella, 2.5 × 10⁴ ± 1 × 10⁴ CFUs). N = 3 with 3 larvae per time point (mean ± SD). (J) Expression of cxcl8a, cxcl18b, il1b, and il10 following reinfection with Shigella in naïve (2.5 × 10⁴ ± 1 × 10⁴ CFUs) and Shigella-trained (2.7 × 10⁴ ± 4.5 × 10³ CFUs) as compared to PBS-injected controls. N = 3 with >5 larvae per time point (mean ± SEM). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, two-way analysis of variance (ANOVA) with Sidak’s multiple comparisons test (C, D, H, I, and J). ns, not significant.