Bulk RNA seq of macrophages isolated from Kmo morphant or control morphant zebrafish larvae infected with <em>Salmonella Typhimurium</em>

The kynurenine pathway of tryptophan degradation has been implicated in various diseases, including cancer, neurodegenerative disorders, and infectious diseases. A key branchpoint in this pathway is production of the metabolite 3-hydroxy-kynurenine (3-HK) by the enzyme kynurenine 3-monooxygenase (Kmo). We recently found that administration of exogenous 3-HK promotes survival to Salmonella Typhimurium infection in zebrafish larvae by restricting bacterial expansion in macrophages via a systemic mechanism that targets kainate sensitive glutamate receptor (KAR) ion channels. Here, we show that endogenous production of 3-HK by Kmo, likewise, is required for defense against systemic Salmonella Typhimurium infection in vivo and that loss of endogenous production of 3-HK impairs macrophage microbicial activity, resulting in increased bacterial expansion. Mechanistically, 3-HK acts by antagonizing KARs to promote lysosome acidification and subsequent control of bacterial burden. Finally, we establish a novel link between activity at KARs and lysosomal acidification in macrophages, revealing a novel regulatory role for KARs in promoting macrophage microbicidal activity and a novel mechanism though which 3-HK promotes control of bacterial infection. Methods Bulk RNA sequencing transcriptomes of macrophages isolated from 3 days post-fertilization zebrafish larvae. Fluorescently labeled cells were isolated in bulk from a single cell suspension obtained by enzymatic digestion of groups consisting of 5 homozygous Tg(mpeg1:mCherry) transgenic animals (7-13 biological replicates/condition) injected with either the translation-blocking kmo morpholino (Kmo MO) or standard control morpholino (Ctrl MO). cDNA libraries were constructed from a range of 50-800 collected cells with an in-house adaptation of the Smart-seq2 protocol in combination with the Nextera XT Library preparation kit (Illumina, Inc., San Diego, CA). Library size and concentration were evaluated using the TapeStation 2200 system (Agilent) and a Qubit fluorometer (Invitrogen) before sequencing. Samples were multiplexed and pair end sequences were generated with NovaSeq SP sequencing system (Illumina) generating 5.5e10^7 reads per sample. Samples were demultiplexed and FASTQ files representing each sample were generated. Any remaining adapter sequences were removed using skewer (0.2.2) and FASTQ files were assessed for quality control using FASTQC (0.11.5). Reads were aligned to the Ensembl zebrafish reference genome GRCz11 using Hisat2 (2.1.0) and counts were quantified using HTSeq-Count (0.12.4). Differential gene expression analysis was performed using DESeq2 (1.38.3). Gene ontology analyses were performed using gene set enrichment analysis (GSEA) and the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses with an FDR below 0.05 using gseKEGG from ClusterProfiler (v4.6.2).