Transcriptional profiling of monocyte-derived MMs predicts their crosstalk with enteric neurons. (A) Percentage of total viable cells (left) and absolute cell counts (right) of major immune cell types in the colonic muscularis single-cell suspensions from 1×DSS- or water-treated Ccr2^RFP/+Cx3cr1^GFP/+ reporter mice at week 1, quantified by FC (n = 5 per group). (B) Percentage of total viable cells (left) and absolute cell counts (right) of MMs and M-T cells in 1×DSS- or water-treated WT mice at week 1, quantified by FC (n = 5 per group). (C) Experimental design used in D and Fig. 4 K: 3×DSS and tamoxifen (Tam) were given as shown. (D) Percentage of tdTomato⁺ cells among Ly6c^hi and Ly6c^lo monocytes, and PMNs in the blood of tamoxifen- and 1×DSS- or water-treated Ccr2^ER-CreR26-STOP^fl/fltdTomato mice, quantified by FC (n = 3 per group). Sampling was done in the same animals across time points. (E) Percentage of MHCII⁺ cells among Ccr2:tdTomato⁺ cells in 1×DSS or water-treated Ccr2^ER-CreR26-STOP^fl/fltdTomato mice, quantified by confocal microscopy of whole muscularis (n = 3 per group). Data represent the mean ± SEM of 5–6 fields of view analyzed per mouse per group. (F) Volcano plot showing a comparison of gene expression between the most distinct Mo-MMs: DSS MM1 and DSS MM4 from the same experiment as in Fig. 4 F. The limma model was used to calculate the P value and fold change. The cutoff for significance is adjusted P value: 1E–5 and Log₂ fold change (Log_2-FC) >1.5 or <−1.5. (G) Heat map showing genes (referred to as MM Core Signature genes) highly expressed in MMs as compared to mucosal macrophages (Muc. Mϕs) in ImmGen gene array dataset generated from normal small bowel MNPs. (H) scRNA-seq dot heatmap showing expression of MM Core Signature genes across five MM clusters identified in Fig. 4 E. Color denotes the average expression level, and size represents the percentage of cells expressing the indicated genes. (I) Subset RNA-seq heatmap showing expression of MM Core Signature genes across MM subsets defined by FC in Fig. 4 F, and isolated by four-way FACS from 1×DSS- or water-treated Ccr2^RFP/+Cx3cr1^GFP/+ mice at week 1. (J) Subset RNA-seq heatmap showing expression of brain microglia–specific genes from the literature by MM subsets defined by FC in Fig. 4 F, isolated by four-way FACS from 1×DSS- or water-treated Ccr2^RFP/+Cx3cr1^GFP/+ mice at week 1. (K) Subset RNA-seq heatmap showing expression of pro- and anti-inflammatory genes by MM subsets defined by FC in Fig. 4 F, isolated by four-way FACS from 1×DSS- or water-treated Ccr2^RFP/+Cx3cr1^GFP/+ mice at week 1. All graphs show the mean ± SEM, n—mouse. Data are representative of at least two independent experiments with similar results (A, B, D, and E). Statistical analyses: unpaired Student’s t test (B, D, and E) and multiple t test (A), *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, and ****P ≤ 0.0001.