Peds1 deficiency promotes a proinflammatory stage with increased neutrophil and macrophage apoptosis.

One-cell stage zebrafish eggs were microinjected with std or peds1 crRNA/Cas9 complexes and the following parameters were evaluated: (A) Larval length and representative images (brightfield channel) at 3- and 5 dpf, (B) Nfkb fluorescent reporter expression levels and representative images (green channel; Tg(NFkB-RE:eGFP)) at 3 and 5 dpf analyzed by fluorescence microscopy, (C) Il1b fluorescent reporter expression levels and representative images (green channel; Tg(il1b:GFP-F)^ump3) at 3 dpf analyzed by fluorescence microscopy. D Transcript levels of the indicated genes analyzed at 3 dpf by RT-qPCR. E, F Number of neutrophils and macrophages and representative images of microinjected larvae after 48 h of treatment with caspase 3 inhibitor (C3I) or vehicle (DMSO) were quantitated by fluorescence microscopy (red channel; Tg(lyz:DsRED2) and Tg(mfap4.1:Tomato), respectively). G, H Hematopoietic stem and progenitor cells (green channel; Tg(-6.0itga2b:eGFP)) and erythrocytes (red channel: Tg(gata1a:dsRED)) number were quantitated by fluorescence microscopy. I, J Number of neutrophils and macrophages and representative images of larvae microinjected to specifically express peds1 in neutrophils using the neutrophil-specific myeloperoxidase (mpx) promoter and analyzed by fluorescence microscopy. Each point represents one larva and the mean ± SEM of each group is also shown. P-values were calculated using one-way ANOVA Tukey’s and multiple range or unpaired Student’s t-test. n.s, not significant, *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.001. auf arbitrary units of fluorescence.

Peds1 deficiency exacerbates chronic skin inflammation.

A Schematic of the experimental procedure used for chronic skin inflammation assays. Spint1a-deficient one-cell stage eggs were microinjected with std or peds1 crRNA/Cas9 complexes and imaged at 3 and 5 dpf. Treatments of interest were added to dechorionated embryos at 1 dpf by bath immersion and renewed daily. Spint1a-deficient larvae showed chronic skin inflammation with increased skin neutrophil infiltration, Nfkb fluorescent reporter expression levels and keratinocyte aggregates. B Analysis and representative images (brightfield channel) of larval length at 3 and 5 dpf. C Number of keratinocyte aggregates in the skin (red arrows) and representative images (brightfield channel) at 3 dpf. D Analysis and representative images of Nfkb fluorescent reporter expression levels (green channel; Tg(NFkB-RE:eGFP)^sh235) at 3 dpf by fluorescence microscopy. E Total number and percentage of neutrophils in the skin and representative merge images (brightfield and red channel; Tg(lyz:DsRED2)^nz50) quantitated by fluorescence microscopy. F Number of skin aggregates and (G) percentage of skin neutrophils quantitated in crstd and crpeds1 spint1a-/- larvae treated for 48 h with 20 µM HsVEPE1, 20 µM HsAEPE1 or vehicle (DMSO). H Representative merge images (brightfield and red channel; Tg(lyz:DsRED2)^nz50) of each treatment analyzed in (F) and (G) by microscopy fluorescence are shown. Each point represents one larva and the mean ± SEM of each group is also shown. P-values were calculated using one-way ANOVA and Tukey’s multiple range or unpaired Student’s t-test, as appropriate. n.s, not significant, *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.001. auf arbitrary units of fluorescence.

Peds1 deficiency impairs inflammation resolution and causes aberrant immune cells recruitment in a sterile tail injury model.

A Schematic of the experimental procedure used for tail injury assays. One-cell stage zebrafish eggs were microinjected with std or peds1 crRNA/Cas9 complexes. At 3 dpf larvae were tail amputated and then inflammation and immune cell recruitment at the injury site (dotted lines) were analyzed at 3, 6, 24 and 48 h post-wounding (hpw) by fluorescence microscopy. The wound area is defined as the region between the amputation edge and the end of the caudal hematopoietic tissue (CHT). B, C Number of neutrophil and macrophages recruited at the injury site and representative merge images (brightfield and red channel; Tg(lyz:DsRED2)^nz50 and Tg(mfap4.1:Tomato)^xt12, respectively). D Number of M1-like macrophages (Tnfa⁺) recruited at the injury site and representative merge images (red channel: Tg(mfap4.1:Tomato)^xt12 and green channel: Tg(tnfa:eGFP-F)^ump5). E Analysis of H₂O₂ release at the wound site using the fluorogenic substrate acetyl-pentafluor-obenzene sulphonyl fluorescein. E–G Analysis and representative images of Il1b (green channel; Tg(il1b:GFP-F)^ump3) and Nfkb fluorescent reporter expression levels (green channel; Tg(NFkB-RE:eGFP)^sh235) at the wound site. Each point represents one larva and the mean ± SEM of each group is also shown. P-values were calculated using one-way ANOVA and Tukey’s multiple range. n.s, not significant, *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.001. auf arbitrary units of fluorescence.

Peds1 deficiency hampers tissue regeneration.

A Schematic of the experimental procedure used for tail injury and regeneration assays. Three dpf larvae previously microinjected with std or peds1 crRNA/Cas9 complexes alone or in combination with cril1b were tail fin amputated and regeneration was evaluated by measuring regenerated fin areas up to 5 dpf (yellow). B, C Measurement of regenerated fin areas and representative images (brightfield channel). D Analysis and representative images of Il1b fluorescent reporter expression levels (green channel; Tg(il1b:GFP-F)^ump3) by microscopy fluorescence. Each point represents one larva and the mean ± SEM of each group is also shown. P-values were calculated using one-way ANOVA and Tukey’s multiple range. n.s, not significant, *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.001. auf arbitrary units of fluorescence.

Peds1 deficiency increased susceptibility to bacterial infection.

A Schematic of the experimental procedure used for infection assays. Single-cell zebrafish embryos microinjected with std or peds1 crRNA/Cas9 complexes were dechorionated and infected at 2 dpf through either the otic vesicle (neutrophil recruitment) or yolk sac (larval survival) with Salmonella enterica serovar Thyphimurium (ST). Treatments of interest were added daily from 1 dpf by immersion and the number of surviving larvae was counted daily for the next 5 days. B Survival analysis of crstd and crpeds1 larvae infected with ST and treated with 20 µM HsVEPE1 or vehicle (DMSO). C Survival analysis of crstd and crpeds1 larvae infected with ST and treated with 20 µM HsAEPE1 or vehicle (DMSO). D Survival analysis of crstd and crpeds1 larvae infected with ST and treated with caspase 3 inhibitor (C3I, 50 µM) or vehicle (DMSO). E, F Number of neutrophil in the otic vesicle and body at 1 and 6 h post-infection in crstd and crpeds1 larvae treated with 50 µM C3I or vehicle (DMSO) counted by fluorescence microscopy (red channel; Tg(lyz:DsRED2)^nz50). G Survival analysis of crstd and crpeds1 larvae injected in combination with antisense (As) or csf3a mRNA and infected with ST. H Number of neutrophil in whole body in uninfected crstd and crpeds1 larvae of 3 dpf treated with 50 µM C3I or vehicle (DMSO) counted by fluorescence microscopy (green channel; (mpx:eGFP)ⁱ¹¹⁴). B–D and G A log-rank test was used to calculate the statistical differences in the survival of the different experimental groups. E, F Each point represents one larva and the mean ± SEM of each group is also shown. P-values were calculated using one-way ANOVA and Tukey’s multiple range. n.s not significant, *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.001.

Peds1a/b DKO line shows developmental delay, neutropenia, monocytopenia and exacerbated inflammation.

A Generation of a DKO line by whole deletion of peds1a and peds1b genes using CRISPR-Cas9. B–F Total plasmalogen and precursor quantification (B), larval length (C), number of neutrophil (D) and macrophage (E), and transcript levels of nfkb1, il1b, tnfa and clxcl8a genes, assayed by RT-qPCR (F). Each point represents one larva and the mean ± SEM of each group is also shown. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001. ROI region of interest, CHT caudal hematopoietic tissue.

Model showing the effect of inhibiting plasmalogen synthesis under basal and different inflammatory conditions.

Inhibition of plasmalogen synthesis promotes a proinflammatory stage with increased neutrophil and macrophage apoptosis. Plasmalogen deficiency also exacerbates chronic skin inflammation, impairs inflammation resolution and tissue regeneration, and results in increased susceptibility to infection.