Fig. 2: Identification of moAMs and temporal analysis of pro-inflammatory genes in AM and IM subsets.

A UMAP Visualization of cell clusters from Naïve and 2-weeks Post-infection timepoint. The NOS2⁺ IMs, NOS2^- IMs and moAMs clusters are highlighted. B Pseudotime analysis of macrophages on the UMAP projection. Key features of the trajectory, including leaves (gray) and branch points (black), are annotated directly on the plot. C DotPlot showing the expression levels of key extravasation and adhesion markers across various macrophage clusters. Dot size and color intensity indicate percentage and average expression level, respectively. D Heatmap of differential gene expression for selected genes between bystander and infected moAMs. Each row represents a gene, and each column represents a cell. N = 1048 cells for moAMs Bystander and n = 1537 cells for moAMs Infected. E (Left Panel): Violin plot showing the aggregate expression level of Nos2 based on GFP status. Expression levels are visualized using violin plots overlaid with jitter plots to show individual data points and mean values represented by solid white points. Statistical significance was assessed using a two-sided Wilcoxon test (p < 2.22e⁻¹⁶). Effect size measured as Cliff’s Delta is also displayed directly on the plot. (Right Panel): UMAPs of infected cells. In the top plot, cells are color-coded based on their Nos2 expression level, while in the bottom plot, they are color-coded based on their GFP status. F UMAP of CD38 staining levels across cell subsets. G Volcano plot showing differential gene expression (DGE) in NOS2⁺ and NOS2⁻ IMs, with key genes of interest labeled. DGE analysis was performed between NOS2⁺ IMs and NOS2⁻ IMs using a two-sided Wilcoxon rank-sum test. P values were adjusted for multiple comparisons using the Bonferroni correction. Genes with an average log₂ fold change >0.58 and adjusted p < 0.05 were classified as significantly upregulated in NOS2+ IMs, while those with a log2 fold change <−0.58 and adjusted p < 0.05 were classified as significantly downregulated. Exact p values and log₂ fold changes for all genes are provided in Supplementary Data 1. Source data are provided as a Source Data file.