Monocyte-derived MMs are the dominant immune cell type expanding in the myenteric plexus in response to colitis. (A) Uniform Manifold Approximation and Projection (UMAP) clustering analysis of scRNA-seq dataset generated from colonic muscularis of normal adult WT mice. Cell type for each cluster annotated based on specific marker genes. ICC, interstitial cells of Cajal; NK cells, natural killer cells. (B) UMAP clustering analysis of all MNPs in the scRNA-seq dataset described in A. DC, dendritic cell. (C) Complementary to Fig. 4 E: normalized gene expression of 12 marker genes across muscularis MNP clusters identified in B. (D) FC t-SNE plots showing immune cell landscape of CD45⁺ viable single cells in colonic muscularis from 1×DSS- or water-treated Ccr2^RFP/+Cx3cr1^GFP/+ reporter mice at week 1. (E) FC plots showing gating strategy for colonic MM subsets and their percentages based on Ccr2-RFP expression in 1×DSS- or water-treated Ccr2^RFP/+Cx3cr1^GFP/+ mice described in D, gated on CD45⁺ viable single cells. (F) FC plots showing colonic MM subsets and their percentages based on Ly6c expression in the same 1×DSS- or water-treated Ccr2^RFP/+Cx3cr1^GFP/+ mice as in D, gated on CD45⁺CD11b⁺CD16/32⁺ viable single cells. (G) Percentage of total viable cells (left) and absolute cell counts (right) of MM subsets based on Ly6c expression in the experiment described in D, quantified by FC (n = 5 per group). (H) Fluorescence microscopy images of colonic myenteric plexus from 1×DSS- (2.25%) or water-treated WT mice at week 1, stained with MHCII (MMs), βIII-tubulin (nerve fibers), and CD3 (T cells) antibodies. White arrowheads point to CD3⁺ T cells. Scale bars, 50 μm. All graphs show the mean ± SEM, n—mouse. Data are representative of at least two independent experiments with similar results (D–H). Statistical analyses: unpaired Student’s t test (G), ****P ≤ 0.0001. MNPs, mononuclear phagocytes.