CD4, Tim-4, and CD163 identify transcriptionally distinct macrophages in the small intestine. (A) Left: Representative flow cytometry plots showing the frequency of Tim-4⁻CD4⁻-, Tim-4⁻CD4⁺-, and Tim-4⁺CD4⁺-expressing cells within the CD163⁻ and CD163⁺ subsets of total small intestinal macrophages from WT mice. Right: Frequency of Tim-4⁻CD4⁻-, Tim-4⁻CD4⁺-, and Tim-4⁺CD4⁺-expressing cells within the CD163⁻ and CD163⁺ subsets of total small intestinal macrophages from WT mice. Data (n = 4 per group) are pooled from two independent experiments. Statistical comparisons were performed with an unpaired t test with Welch’s correction for parametric data and a Mann–Whitney test for nonparametric data. *, P ≤ 0.05; **, P ≤ 0.01. (B) Principal component analysis (PCA) of global gene expression from CD163⁻ and CD163⁺ subsets of Tim-4⁻CD4⁻, Tim-4⁻CD4⁺, and Tim-4⁺CD4⁺ macrophage of the small intestine isolated by FACS from five pooled WT mice, from at least three independent sorts. (C) Gene expression profile of the 3,206 genes differentially expressed (P-adjusted <0.001) in CD163⁻ and CD163⁺ subsets of Tim-4⁻CD4⁻, Tim-4⁻CD4⁺, and Tim-4⁺CD4⁺ macrophage of the small intestine with clusters identified by k-means clustering. (D) Top GO terms associated with each of the four clusters formed by the 3,206 DEGs. (E) Heatmaps showing expression profiles of genes in the top two pathways identified by PANTHER pathway analysis for clusters 1 and 2.