Fig. 2: irg1-KI:GFP knock-in reporter enables isolation and characterization of macrophage subpopulations based on activation states.

a irg1-KI:GFP reporter brightly labels auto-inflammatory macrophages in nlrc3l mutants, enabling rapid and reliable sorting of live nlrc3l mutants from siblings using a low-magnification stereoscope. b Schematic for FACS sorting of macrophages from nlrc3l mutants and siblings with irg1-KI:GFP, isolating subpopulations based on GFP levels (high, low, negative). Created in BioRender. Shiau, C. (2026) https://BioRender.com/f3hmn0s. c Contour plots from FACS sorting showing the frequency of macrophage subpopulations with varying irg1 levels (based on GFP expression). d Histogram depicting irg1 expression levels inferred from GFP intensity in macrophages across different genotypes or conditions (showing four independent samples per category). Auto-inflammatory nlrc3l mutants exhibit higher irg1 levels compared to LPS-activated controls. The irg1-low and irg1-high regions are color-coded. e Four-way Venn diagram comparing macrophage populations from bulk RNA-seq analysis to identify genes enriched in nlrc3l mutant subpopulations versus control macrophages. The color-coded shapes represent the gene sets that were compared to acquire the list of upregulated genes in each category. f Bar graphs of enriched biological pathways from Metascape analysis in irg1-high mutant, irg1-low mutant, and control macrophage populations. g Heat maps show RNA-seq expression levels of selected genes and immune markers across macrophage populations. Mutant macrophages are enriched in inflammatory and stress response genes, while irg1-high and irg1-low mutant macrophages show differential expression in vesicle/transport and ECM/immune categories, respectively. mrc1b/cd206 as highlighted in red text is especially downregulated in all mutant macrophages. h Heat map showing unsupervised hierarchical clustering analysis with NG-CHM, revealing shared and unique biological pathways significantly enriched in mutant macrophage subpopulations compared to control macrophages. i In vivo imaging of irg1-KI:GFP and tnfa:GFP in nlrc3l mutants and their control siblings at baseline reveals elevated irg1 and induced tnfa expression in mutant macrophages (mpeg1 + ), which are morphologically altered and vesicle-filled, and accompanied by abnormal irg1 expression in mutant neutrophils (lyz + ) (demarcated by dotted lines and asterisks). The inset shows a single channel for mpeg1:BFP or tnfa:GFP as indicated. Arrows, indicator of macrophage; asterisks, indicator of neutrophils. All scale bars show 25 um. f, h DEGs analyzed for pathway enrichment were derived from DESeq2 analysis using default Wald test to obtain p-values corrected for multiple testing.