Neutrophil-specific miR-99 overexpression hinders neutrophil recruitment and motility. (A) Schematic of the Tol2-lyzC:miR-99/Dendra2 plasmid, injected into wild-type AB zebrafish embryos to generate the transgenic line Tg(lyzC:miR-99/Dendra2) (miR-99) and Tg(lyzC:Dendra2).(B) The relative expression level of miR-99, miR-223, and let-7e (normalized to U6 expression) in vector and miR-99 lines determined by RT-qPCR, mean ± s.d. (N = 3 biological replicates with ten larvae at each time point in each group). (C, D) Representative images and quantification of total neutrophils in vector and miR-99 lines. Scale bar: 500 μm. (E, F) Representative images and quantification of neutrophil recruitment to tail transection site in vector or miR-99 larvae at 1-hour post-injury. Neutrophils in the boxed region were quantified. Scale bar: 100 μm. (G, H) Representative images and quantification of neutrophil recruitment to localized ear infection in vector or miR-99 larvae at 1-hour post-infection. The infected ear is indicated with the circle. Scale bar: 100 μm (I, J) Tracks and quantification of neutrophil motility in vector or miR-99 larvae. Scale bar: 50 μm. All experiments were performed with F2 larvae at three dpf. One representative result of three independent experiments was shown (n=20). P-value was calculated with unpaired Student’s t-test.

MIR-99 overexpression inhibits chemotaxis of dHL-60 cells. (A) Construct for inducible miRNA expression. The miRNA and a dendra2 reporter (green) are controlled by TRE, Tetracyclin response element (red). The PGK promoter drives constitutive expression of puromycin resistance gene (cyan) and the rtTA, reverse tetracycline-controlled transactivator (red). (B) Quantification of MIR-99, MIR-233, and LET-7E in dHL-60 cell lines with/without induced expression of the vector control or MIR-99. The result from three independent experiments is normalized to U6 expression levels and presented as mean ± s.d., students’ t-test. (C) Cell populations gated for downstream cell profile analysis and transwell quantification. (D) Doxycycline-mediated induction in the vector or miR-99 expressing dHL-60 lines. Cells without doxycycline-mediated induction were used as a baseline. Percentage of cells with dendra2 levels above the baseline are shown. (E) Surface CD11b levels in vector or miR-99 expressing dHL-60 cells. Undifferentiated cells or differentiated cells stained with an isotype control were used as a baseline. Percentage of cells with CD11b levels above the baseline with or without doxycycline-mediated induction are shown. (F) Annexin V staining in the total cell population (excluding debris) of vector or miR-99 expressing dHL-60 cells. An unstained live sample is used to determine the baseline. Percentage of cells with AnnexinV levels above the baseline with or without doxycycline-mediated induction are shown. (C–F) One representative experiment of three independent trials is shown. (G–J) (G) Transwell migration of dHL-60 cells with/without induced expression of the vector control or MIR-99 toward fMLP, (H) phagocytosis of BioParticles by dHL60 cells represented as mean FITC, (I) H₂O₂ concentration in PMA-induced dHL60 cells, and (J) Quantification of dHL60 undergoing NETosis (left) and representative images (right). Scale bar, 100µm. Results are presented as mean ± s.d., from three independent experiments and normalized to vector, Kruskal–Wallis test or Mann-Whitney test.

miR-99 overexpression suppresses the expression of roraa in zebrafish neutrophils. (A) Volcano blot of DEGs with significant expression changes in miR-99 expressing neutrophils. Blue: down-regulated differentially expressed genes (DEGs); Magenta: up-regulated DEGs. (B) Significantly altered pathways in the down-regulated DEGs. (C) Alignment of miR-99-1-5p and predicted miR-99 binding sites in zebrafish roraa 3’UTR. (D) Transcript level quantification of predicted downregulated genes in neutrophils sorted from the vector or miR-99 zebrafish line. Results from three independent experiments are normalized to rpl32 and presented as mean. Holm-Sidak test. (E) Suppression of Renilla luciferase expression via binding to zebrafish roraa 3’UTRs by miR-99. Results from three independent experiments are normalized to firefly luciferase and presented as mean ± s.d., Kruskal–Wallis test.

Pharmacological inhibition of Rora reduces neutrophil motility and chemotaxis in zebrafish and humans. (A, B) Tracks and quantification of neutrophil motility in zebrafish larva treated with SR3335 (100μM), SR2211 (100μM), or VPR66 (25μM). Scale bar, 200 µm. Three embryos, each from three different founders, were imaged. Quantification of neutrophils in one representative video is shown, Kruskal–Wallis test. (C, D) Representative images and quantification of neutrophils recruited to the infected ear in zebrafish larva treated with RORα specific inhibitor (SR3335, 100μM), RORγ specific inhibitor (SR2211, 100μM) or pan-ROR family inhibitor (VPR66, 25μM). Scale bar, 100 µm. (E, F) Representative images and quantification of neutrophils recruited to tail fin transection sites in zebrafish larva treated with SR3335 (100μM), SR2211 (100μM), or VPR66 (25μM). Scale bar: 500 µm. (C–F) The result from one representative experiment is shown as mean, Mann–Whitney test. (G, H) Representative tracks and mean velocity of primary human neutrophils treated with SR3335 (50μM), SR2211 (50μM), or SR1001 (pan-ROR family inhibitor) (50 µM) migrating towards fMLP in 3D matrigel. Scale bar, 100 µm. Representative results for three individual trials are shown. The result is presented as mean, Mann–Whitney test. (I) Neutrophil recruitments after zebrafish tail wounding at different dosages of SR3335 treatment compared to 1% DMSO treatment, Kruskal–Wallis test. (J) Transwell migration of primary human neutrophils treated with DMSO (0.1%) or SR3335 at 10, 30, or 100 μM toward 100 nM fMLP. Results are presented as mean ± s.d., from three independent experiments and normalized to DMSO (0.1%), Kruskal–Wallis test.

Dominant-negative Rorα suppresses neutrophil motility and chemotaxis. (A) Schematic of the construct for neutrophil-specific expression of vector control or rorα dominant-negative (DN). (B, C) Representative images and quantification of total neutrophil numbers in vector or rorα DN zebrafish line. Scale bar: 500 μm. (D, E) Representative images and velocity of random neutrophil migration in vector or rorα DN zebrafish line. Scale bar, 100 µm. Three embryos each from three different founders were imaged, and quantification of neutrophils in one representative video is shown, Kruskal–Wallis test. (F, G) Representative images and quantification of neutrophils recruited to the infected ear in vector or rorα DN zebrafish line. Scale bar, 100 µm. (H, I) Representative images and quantification of neutrophil recruitment to the tailfin transection sites in vector or rorα DN zebrafish line. Scale bar, 200 µm. The result is presented as mean, Mann–Whitney test. (J) Simultaneous imaging of utrophin-GFP distribution in neutrophils expressing either mCherry alone or with Rora DN. Data are representative of more than three separate time-lapse videos. Scale Bar, 50 µm. (B, C, F–I) Representative results for three individual trials are shown. (K) Schematics of the plasmid constructs for neutrophil-specific Cas9 expression (upper construct) and for ubiquitous expression of sgRNAs with a neutrophil-specific expression of GFP (lower construct). (L, M) Representative images (L) and quantification (M) of neutrophil motility at head region of 3dpf larvae from Tg(LyzC: Cas9, Cry: GRFP, U6a/c: rora guides, LyzC: GFP) fish. Student’s t-test. Scale bar, 100 µm. (N) Deep sequencing of the rora loci targeted by guide RNAs in (K). The sequences on the top are wild-type sequences, and the five most frequent types of mutations are shown. Point mutations, deletions, and insertions are all observed.

Rorα in neutrophils protects zebrafish against Pseudomonas aeruginosa infection. (A) Survival curve of 3 dpf larvae from the vector or miR-99 line after i.v. infection with Pseudomonas aeruginosa (PAK). (B) Survival curve of 3 dpf larvae from the mcherry or roraa DN line larvae after i.v. infection with PAK. (C) Survival curve of 3 dpf larvae treated with DMSO or SR3335 (100µM) injected with PAK. (D) Survival curve of 3 dpf larvae treated with DMSO or SR2211 (100µM) injected with PAK. One representative experiment of three independent biological repeats (n = 20 each group) is shown. The result is analyzed with Gehan–Breslow–Wilcoxon test.

RNA-Seq reveals the direction of rorα possible downstream genes in neutrophil migration. (A) Volcano blot of DEGs with significant expression changes in rorα DN neutrophils. Cyan: down-regulated differentially expressed genes (DEGs); Magenta: up-regulated DEGs. (B) Significant gene function enrichment of human homologs of genes in the down-regulated DEGs. The heat map indicates the log2 fold change of the selected DEGs. The relevance of individual DEGs in each pathway is indicated with purple. (C) Venn diagram showing human homologs of genes in the down-regulated DEGs overlapping with putative RORα target genes in THP-1 or HUVEC cells. (D) Transcript level validation of downregulated genes in neutrophils sorted from rorα DN line zebrafish. Results from three independent experiments are normalized to rpl32 and presented as mean ± s.d., students’ t-test.