Antimicrobial activity of phosphatidylserine/phosphatidic acid (PS/PA) liposome prophylactic administration in wild-type (WT) and cftr-LOF zebrafish embryos. (A) Schematic representation of PS/PA liposome prophylactic treatment. Zebrafish embryos were treated with PS/PA liposome at 28 hpf, then systemically or locally infected with 100–300 CFU PAO1-GFP at 48 hpf and analyzed for bacterial burden at 8 or 20 h post infection. (B) Bacterial load (relative CFU/embryo) in systemically infected WT and cftr-LOF embryos, control (ctrl), and PS/PA liposome treated at 8 hpi. Results are presented as mean ± SEM. (C) Representative images of PAO1-GFP systemic bacterial infection in ctrl and PS/PA liposome treated embryos. (D,E) Quantitative analysis (fluorescence pixel count) of PAO1-GFP locally injected in the close cavity of the hindbrain ventricle of WT and cftr-LOF embryos, control (ctrl) and PS/PA liposome treated. The mean and the min to max values of at least two independent experiments (3–10 embryos/treatment) were reported on floating bars. (F) Representative images of PAO1-GFP ventricle bacterial infection in ctrl and PS/PA liposome treated embryos. Statistical significance was assessed by unpaired Student’s t test: **p < 0.01; *p < 0.05. Scale bar indicates 500 μm in panel (C) and 200 μm (dorsal) and 150 μm (lateral) in panel (F).

Macrophage activation in wild-type (WT) and cftr-LOF zebrafish embryos upon phosphatidylserine/phosphatidic acid (PS/PA) liposome prophylactic administration. (A) Schematic representation of PS/PA liposome prophylactic treatment. Zebrafish embryos were treated with PS/PA liposome at 28 hpf, then locally infected with PAO-GFP at 48 hpf and analyzed for macrophage activation at 6 h post infection. (B) Representative image of macrophage migration toward the PAO1-GFP bacteria injected in the hindbrain ventricle (circle area) of ctrl or PS/PA liposome treated cftr-LOF embryos. (C,D) Quantification of mpeg1:mcherry positive macrophages in the selected area of the ventricle of ctrl or PS/PA liposome treated WT (C) or cftr-LOF embryos (D). (E) Representative image of red macrophages of the Tg(mpeg1:mcherry) embryos phagocyting PAO1-GFP bacteria (arrowheads), injected in the hindbrain ventricle (visual imaging in the right-upper box). (F,G) Quantitative analysis (Log₁₀ fluorescence pixel count, related to colocalization area) of phagocytic activity of macrophages against PAO1-GFP bacteria in WT (F) and cftr-LOF embryos (G), control (ctrl) and PS/PA liposome treated. (H,I) Pro- and anti-inflammatory cytokines expression by RT-qPCR analyses at 20 hpi in WT (I) and cftr-LOF embryos (J), ctrl and PS/PA liposome treated embryos, systemically infected with PAO1. (J) Bacterial load quantification (relative CFU/embryo) at 8 hpi in ctrl and PS/PA liposome treated WT embryos treated with Lipo-clodronate. Unpaired Student’s t test: ***p < 0.001; *p < 0.05; ns: not significant. Data resulted from at least two (C,D,F,G) or three (H–J) independent experiments and results are presented as mean ± SEM. Scale bar indicates 200 μm in panel (B) 20 μm in panel (E).

Antimicrobial activity in WT and cftr-LOF zebrafish embryos of the combination treatment with phosphatidylserine/phosphatidic acid (PS/PA) liposome in prophylactic administration and phage therapy. (A) Schematic representation of PS/PA liposome prophylactic treatment followed by systemic bacterial infection and CKΦ-administration. Zebrafish embryos were treated with PS/PA liposome at 28 hpf, then systemically infected with PAO1-GFP at 48 hpf, treated with CKΦ 3 h later, and analyzed for bacterial burden at 8 or 20 h post infection. (B,C) Bacterial load (relative CFU/embryo) in systemically infected wild-type (WT) (B), and cftr-LOF embryos (C), control (ctrl), PS/PA liposome, CKΦ and PS/PA liposome + CKΦ treated. Statistical significance was assessed by One-way ANOVA followed by Tukey’s post hoc test: ***p < 0.001; *p < 0.05; ns, not significant. Data resulted from three independent experiments and results are presented as mean ± SEM.

Combination treatment with phosphatidylserine/phosphatidic acid (PS/PA) liposome and CKΦ in therapeutic administration elicits synergistic effect in antimicrobial activity in wild-type (WT) and decreases phage-resistant PAO1 proliferation in cftr-LOF zebrafish embryos. (A) Schematic representation of combined administration of PS/PA liposome/CKΦ. 48 hpf zebrafish embryos were systemically infected with phage sensitive (PAO1) and/or resistant PAO1 (PAO1-217-2a-GFP) strains, treated with single or combined PS/PA liposome CKΦ 3 h later and analyzed for bacterial burden at 8 or 20 h post infection. (B,C) Bacterial load (relative CFU/embryo) in systemically infected WT (B) and cftr-LOF embryos (C), control (ctrl), PS/PA liposome, CKΦ and PS/PA liposome-CKΦ treated at 8 hpi. (D–G) Bacterial load (relative CFU/embryo) (D,F) and percentage of PAO1-217-2a-GFP colonies (E,G) in embryos systemically infected with 50% phage-sensitive PAO1 (non-GFP) and 50% phage-resistant PAO1-GFP bacterial suspension. WT (D,E) or in cftr-LOF embryos (F,G), control (ctrl), PS/PA liposome, CKΦ and PS/PA liposome-CKΦ treated. (H) Representative image of phage-sensitive PAO1 and phage resistant PAO1-217-2a-GFP colonies derived from the plating of homogenized infected embryos. Statistical significance was assessed by One-way ANOVA test followed by Tukey’s post hoc correction:***p < 0.001; **p < 0.01; *p < 0.05; ns, not significant. Data resulted from three independent experiments and results are presented as mean ± SEM.

PS/PA liposomes and CKΦ administration does not elicit toxic effects on human CF cells. (A) Cell viability, evaluated by MTT assay, in immortalized human bronchial epithelial cells CuFi-1 expressing F508del CFTR control, treated with phosphatidylserine/phosphatidic acid (PS/PA) liposome, CKΦ or combination of PS/PA liposome-CKΦ for 48 h. (B) Cell viability, evaluated by MTT assay, in THP-1 activated macrophage-like cells treated or not with the CFTR inhibitor. Effects of single or combined PS/PA liposome and CKΦ-administration were evaluated after 48 h of treatment. Unpaired Student’s t test: *p < 0.05; ns, not significant. Results are presented as mean ± SEM.