Fig. 5

Inflammasome and cathepsin-B inhibition decrease ChA-induced myeloid cell infiltration. A–D: Zebrafish larvae were fed for 10 days with normal, 4% FC-enriched, or 3% ChA-enriched diets, and macrophages were isolated by fluorescence-activated cell sorting (FACS). A: Representative confocal images of macrophages stained with BODIPY 493/503 to visualize neutral lipids. B: Quantification of neutral lipid accumulation on isolated macrophages. C: Representative confocal images of macrophages stained with LysoTracker to visualize lysosomes/acidic organelles. Scale bars represent 5 μm. D: Quantification of the lysosome area on isolated macrophages. In B and D, the results are the mean ± SEM of two independent experiments (at least 10 cells were analyzed per condition); ∗∗∗P < 0.001 using one-way ANOVA with Dunnett’s multiple comparisons test. E: In vivo lysosomal imaging of macrophages (m-Cherry) from larvae fed with different diets. Lysosomes were stained with LysoTracker (green). Scale bars represent 5 μm. F–H: Effect of caspase 1 (Casp-1) and cathepsin B (Cath B) inhibitors on neutrophils and macrophage infiltration into the caudal vein of Tg (mpeg.mCherryCAAX SH378, mpx:EGFP i114) zebrafish larvae fed with normal, 10% FC-enriched, or 14% ChA-enriched food. F: Representative confocal images. Images are Z-stacks of fluorescent green (neutrophils) and red (macrophages) cells and the respective bright fields. Dashed lines delineate the caudal vein. Scale bars represent 20 μm. G and H: Quantification of Casp-1 and Cath B inhibitors on neutrophil (G) and macrophage (H) infiltration into the caudal vein. Three independent experiments were performed, and at least three larvae were analyzed per experiment (total of >10 larvae per condition); ∗P < 0.05; ∗∗∗P < 0.001 using two-way ANOVA with Dunnett’s multiple comparisons test.