VD₃ promotes the granulopoiesis in zebrafish larvae. Zebrafish larvae at 2 dpf were exposed to 100 nM VD₃ for 2 days, GZM was replaced at 4 dpf, and 100 nM VD₃ were added simultaneous. a The merged images of Tg(mpx:gfp) zebrafish (6 dpf) showed the abundance and localization of GFP⁺ neutrophils. White dashed line indicates the intestine. b The size of neutrophil population was represented by neutrophil unit as described in Materials and Methods. Graph shows quantification of neutrophil units in whole zebrafish larva at 6 dpf. c Enumeration of intestinal-associated GFP⁺ cells in 6 dpf larvae. d The gene expression by qRT-PCR of csf3a, csf3b, mpx, lysc, and mmp9 from 6 dpf zebrafish larvae treated with 0 or 100 nM VD₃ for 4 days (results are combined from 2 independent experiments, n = 4 replicates/group/experiment, 10–20 larvae/replicate). Data are represented as mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001. VD₃, vitamin D₃; dpf, days post fertilization; mpx, myeloperoxidase; csf3a, colony-stimulating factor 3a; csf3b, colony-stimulating factor 3b; lysc, lysozyme C; mmp9, matrix metalloproteinase9; GZM, gnotobiotic zebra­fish medium.

VD₃ contributes to the granulopoiesis in zebrafish intestine. a The gene expression of csf3a, csf3b, mpx, lysc, and mmp9 in the gut of zebrafish (35 dpf) fed 0 or 800 IU VD₃/kg diets for 2 weeks (n = 12 replicates/group, 2–3 zebrafish/replicate). b Transcript levels of csf3a, csf3b, mpx, lysc, and mmp9 in the gut of WT and cyp2r1 mutant zebrafish in 3 months (n = 8 replicates/genotype). Data are represented as mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001. csf3a, colony-stimulating factor 3a; csf3b, colony-stimulating factor 3b; lysc, lysozyme C; mmp9, matrix metalloproteinase9; dpf, days post fertilization; VD₃, vitamin D₃; WT, wild-type; mpx, myeloperoxidase.

Microbiota impact on granulopoiesis in zebrafish. a–c The gene expression of csf3b (a), mpx (b), and mmp9 (c) in CONVED or GF zebrafish larvae (6 dpf) treated 0 or 100 nM VD₃ for 4 days (results are combined from 2 independent experiments, n = 12 replicates/group, 10–20 larvae/replicate). d–f WT and cyp2r1 mutant zebrafish in 3 months were treated with antibiotics for 1 week, and the gene expression of csf3b (d) mpx (e), and mmp9 (f) transcripts in the gut was analyzed (n = 8 replicates/genotype). Two-way ANOVA with Sidak test was used to test significance. Data are represented as mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001. csf3b, colony-stimulating factor 3; mmp9, matrix metalloproteinase9; dpf, days post fertilization; VD₃, vitamin D3; WT, wild-type; CONVED, conventionalized; GF, germ-free; mpx, myeloperoxidase.

VD₃ enhances neutrophil recruitment to the wound site of amputated fish. a–c The caudal fin of 6 dpf Tg(mpx:gfp) zebrafish. Imaging and quantification of GFP⁺ neutrophils recruited to tail wound at 4 hpw (n = 4/group) (b). d qRT-PCR analysis of IL-8 transcripts in 6 dpf zebrafish larvae treated 0 nM, 10 nM, or 100 nM VD₃ for 4 days, with and without caudal fin amputation (n = 6 replicates/group, 6–15 larvae/replicate). Two-way ANOVA with Sidak test was used to test significance. For c, statistical comparisons were performed within each time point. Data are represented as mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001. dpf, days post fertilization; VD₃, vitamin D₃; WT, wild-type; hpw, hours post wounding; mpx, myeloperoxidase.

VD₃ restrains pathogen infection in zebrafish. a–c Transcript levels of mpx (a), mmp9 (b), and csf3b (c) in unstimulated and E. tarda-exposed zebrafish larvae, pretreated with 0 or 100 nM VD₃ (n = 4 replicates/group, 10–20 larvae/replicate). Two-way ANOVA with Sidak test was used to test significance. d Zebrafish larvae at 3 dpf were immersed with 1 × 10⁸ CFU/mL E. tarda. After 24, 48, and 72 h, the bacteria burden in the larvae was counted (n ≥ 24 larvae/group/experiment, 3 independent experiments). e Zebrafish larvae at 3 dpf were pretreated with control buffer or VD₃ (50 nM) for 48 h. Afterward, the larvae were microinjected with E. tarda (approximately 200 bacteria/larva). The survival of the larvae in each group was recorded up to 36 hpi (n = 10 larvae/group/experiment, 3 independent experiments). Statistical analysis was conducted by Log-rank test. Data are represented as mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001. csf3b, colony-stimulating factor 3b; mmp9, matrix metalloproteinase9; dpf, days post fertilization; VD₃, vitamin D₃; E. tarda, Edwardsiella tarda; hpi, hours post infection; mpx, myeloperoxidase.

Neutrophils are required for VD₃-mediated control of bacterial growth in zebrafish. a Embryos microinjected with control buffer or sgRNAs targeting csf3r were collected at 6 dpf. Genotyping of csf3r amplicons was analyzed by qRT-PCR, and transcript levels of mpx were also tested. b Zebrafish larvae at 5 dpf were microinjected with E. tarda (approximately 200 bacteria/larva). The survival of the larvae in each group was recorded up to 25 hpi (n = 10 larvae/group/experiment, 2 independent experiments). Statistical analysis was conducted by Log-rank test. c–f Zebrafish larvae were immersed with 1 × 10⁸ CFU/mL E. tarda at 3 dpf. After 24, 48, and 72 h, the bacteria burden in the larvae was counted (n ≥ 14 larvae/group/experiment, 2 independent experiments). Data are represented as mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001. dpf, days post fertilization; VD₃, vitamin D₃; E. tarda, Edward-siella tarda; hpi, hours post infection; mpx, myeloperoxidase; WT, wild-type.

Schematic model of the regulation of granulopoiesis and neutrophil functions by VD₃ in zebrafish. ① In adult zebrafish, the gene expression of csf3b and the neutrophil markers were enhanced in the intestine of the fish fed with VD₃-containing diet, compared to those fed with non-VD₃ diet. ② The gene expression of csf3b and neutrophil abundance in the intestine of adult zebrafish with a cyp2r1 mutant were reduced. Interestingly, microbiota is involved in VD-mediated granulopoiesis in adult zebrafish. ③ Addition of exogenous VD₃ promoted granulopoiesis in zebrafish larvae. However, VD-regulated neutrophil generation independent of the microbiota during early development. ④ VD₃ treatment significantly decreased bacterial counts and mortality in zebrafish larvae infected with E. tarda in a neutrophil-dependent manner. ⑤ VD₃ augmented the gene expression of IL-8 and neutrophil recruitment to the site of caudal fin amputation. csf3b, colony-stimulating factor 3b; VD_3, vitamin D₃; E. tarda, Edwardsiella tarda.