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Kmo inhibition suppresses lysosomal acidification in macrophages during infection which is rescued by 3-HK. (A) Schematic of bulk RNA sequencing measuring transcriptional changes in naïve, exposed and infected macrophages from infected kmo and control morphant larvae. Tg(mpeg1.1:mCherry) kmo or control morphant larvae were infected with S. Typhimurium-GFP or left uninfected and mCherry+ macrophages were sorted from infected and naïve fish at 4 HPI. Macrophages from infected fish were further sorted into infected (GFP+) and exposed (GFP-) populations. (B-C)kmo depletion suppresses expression of gene signatures of lysosomal acidification in infected macrophages using GSEA (B, KEGG terms). (C) GSEA plot showing downregulation of the lysosomal KEGG pathway in infected kmo macrophages compared to infected control macrophages. The running enrichment score (green line) demonstrates pathway suppression as genes predominantly cluster near the bottom of the ranked gene list. Vertical black bars mark the positions of pathway genes within the red-blue color gradient which indicates relative gene expression from upregulated (red) to downregulated (blue). NES, normalized enrichment score. FDR, false discovery rate. (D, E, F) Endogenous and exogenous 3-HK promote lysosomal acidification in infected macrophages. Tg(mpeg1.1:mCherry) larvae were infected with S. Typhimurium-GFP and treated with DMSO control or 50µM Ro61-8048 with or without 100µM 3-HK. Embryos were bathed in Lysotracker to stain acidic compartments at 4 HPI and maximum Lysotracker fluorescence intensity was measured using confocal microscopy. Lysotracker intensity was quantified from imaged individual macrophages in the caudal hematopoietic tissue. Data are representative of 3 experiments. (D,F; Values are individual macrophages from 8-13 biological replicates; All data points shown; Unpaired t-test. ****p < 0.0001). (E) Quantification of the percentage of total bright Lysotracker+ macrophages in whole larvae via flow cytometry in 50µM Ro61-8048 and DMSO (vehicle) control treated fish. (Each value represents pooled 5 larvae; Unpaired t-test, ***p < 0.001). Data are representative of 2 experiments. GSEA, gene set enrichment analysis. ctrl, control morphant. kmo, kmo morphant. Ro61-8048, kmo inhibitor. Fig 3A created with Biorender [22].
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