CCR2 ⁺ monocyte-derived MMs expand in the myenteric plexus in response to colitis. (A) Confocal images of colonic myenteric plexus isolated from 3×DSS- or water-treated mice (as in Fig. 1 G but with MHCII channel ON). Images are representative of control (n = 5–10) and DSS (n = 4–13) for each time point. Scale bars, 100 μm. (B) Total MHCII⁺ area in the colonic myenteric plexus shown in A, based on confocal microscopy. Data are shown as the mean value per mouse (left, control, n = 5–10; DSS, n = 4–13) or as distribution of pooled values generated from analysis of 4–9 fields of view per mouse (right). (C) Percentage of total viable cells (left) and absolute cell counts (right) of colonic MMs from mice described in A, quantified by FC (control, n = 5–7; DSS, n = 4–7). (D) Predicted developmental trajectory of five scRNA-seq MM clusters in normal colon determined by slingshot analysis, shown as pseudotime (left) and MM clusters (right). The scRNA-seq dataset is described in Fig. S4. (E) scRNA-seq dot heatmap showing expression of 11 marker genes across five MM clusters displayed in D. Color denotes the average expression level, and size represents the percentage of cells expressing the indicated genes. (F) FC t-SNE plots showing MM subsets of colonic muscularis single-cell suspensions from water- or 1×DSS-treated (2.75%) Ccr2^RFP/+Cx3cr1^GFP/+ reporter mice at week 1 (gated on CD45⁺CD11b⁺CD16/32⁺ viable cells). (G) MFI heatmap showing expression of five MM-specific protein markers in the same FC experiment as in F. (H) RNA-seq heatmap showing expression of 11 marker genes by MM subsets identified in E, isolated by four-way FACS from 1×DSS- or water-treated Ccr2^RFP/+Cx3cr1^GFP/+ mice at week 1 in the same experiment as in F and G. (I) Percentage of total viable cells (left) and absolute counts (right) of colonic MM subsets shown in F–H, quantified by FC (n = 5 per group). (J) Confocal images of colonic myenteric plexus from 1×DSS- or water-treated Ccr2^RFP/+Cx3cr1^GFP/+ mice at week 1, stained with antibodies to GFP (CX3CR1⁺ cells), RFP (CCR2⁺ cells), and βIII-tubulin (nerve fibers). Scale bars, 50 μm. (K) Percentage of monocyte-derived tdTomato⁺ cells among colonic MM subsets and PMNs from tamoxifen- and 1×DSS- (3%) or water-treated Ccr2^ER-CreR26-STOP^fl/fltdTomato mice at week 5, quantified by FC (n = 3 per group). (L) Wheel plots showing top ligand–receptor pair interactions between colonic myenteric neurons and monocyte-derived MM1 (left) or MM4 (right) clusters predicted by CellChat analysis of the scRNA-seq dataset described in Fig. S4. All graphs show the mean ± SEM, n—mouse. Data in A–C, F, G, and I are representative of at least three independent experiments, and data in J and K are representative of at least two independent experiments with similar results. Statistical analyses: unpaired Student’s t test (I), multiple t test (C), and two-way ANOVA for multiple comparisons (B), *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, and ****P ≤ 0.0001. MFI, mean fluorescence intensity.