Enteric neuron-derived CCL2 facilitates pathological myenteric plexus remodeling and postinflammatory GI dysmotility by excessive recruitment of monocytes in response to colitis. (A) Two-dimensional projections of three-dimensional two-photon images of colonic myenteric plexus from 1×DSS- (3%) or water-treated mice, stained with MHCII (MMs) and Hu (neuronal somas) antibodies. Images are representative of control (n = 3) and DSS (n = 1–3) mice for each time point. Scale bars, 100 μm. (B) Myenteric neurons, MMs, their interactions, and neuronal uptake by MMs quantified by two-photon microscopy in the experiment described in A (control, n = 3; DSS, n = 1–3). Data are shown as the mean of pooled values generated from analysis of 5–18 fields of view per group. (C)Ccl2 expression by qPCR in CD45^–CD90⁺CD24⁺ myenteric neurons and other CD45^– stromal cells FACS-purified from colonic muscularis of 1×DSS- or water-treated mice at week 1, normalized to the housekeeping gene Actb (control, n = 1–3; DSS, n = 2, where n = independent cell sorting). (D) Confocal images of colonic myenteric plexus from 1×DSS (2.5%)- or water-treated Ccl2-RFP^fl/fl and WT mice at week 1, stained with MHCII (MMs), tdTomato (Ccl2-RFP), and Hu (neuronal somas) antibodies. White arrowheads indicate CCL2⁺Hu⁺ neurons. Scale bars, 50 μm. (E) Experimental groups used in F–J, analyzed at weeks 1 and 11 after starting DSS. (F) Percentage of total viable cells (left) and absolute cell counts (right) of colonic Mo-MMs in tamoxifen- and 3×DSS-treated (2.15%) SLICK-H^ER-CreCcl2^fl/fl (Cre⁺) and Ccl2^fl/fl littermates (Cre^–), quantified by FC (DSS Cre^– and Cre⁺, n = 8 per group). Baseline shows the mean value for tamoxifen- and water-treated Ccl2^fl/fl littermates (Water Cre^–, n = 5–6). P values above each column indicate statistical differences against baseline. (G) Structural changes in the colonic myenteric plexus of mice described in F, showing counts of neuron clusters, counts of Hu⁺ neurons per cluster, and counts and area of IGFT regions, quantified by confocal microscopy (DSS Cre^– and Cre⁺, n = 7–8 per group; Water Cre^– baseline, n = 4–6). Data are shown as distributions of pooled values generated from analysis of 5 fields of view per mouse. (H) GI transit in tamoxifen- and 3×DSS-treated Cre^– and Cre⁺ littermates at week 11 after starting DSS (DSS Cre^– and Cre⁺, n = 13–14 per group; Water Cre^– baseline, n = 9). P values above each column indicate statistical differences against baseline. (I) Counts of CC7⁺ANNA-1⁺ colonic myenteric neurons analyzed at week 1, quantified by confocal microscopy (DSS Cre^– and Cre⁺, n = 4 per group; Water Cre^– baseline, n = 2). Data are shown as a distribution of pooled values generated from analysis of 5–6 fields of view per mouse. (J) Interactions (contacts) between MHCII⁺ MMs and myenteric Hu⁺ neurons analyzed at week 1, quantified by confocal microscopy (DSS Cre^– and Cre⁺, n = 7–8 per group; water Cre^– baseline, n = 4). Data are shown as distributions of pooled values generated from analysis of 5 fields of view per mouse. All graphs show the mean ± SEM, n—mouse, unless stated differently. Data are representative of at least two independent experiments with similar results. Statistical analyses: unpaired Student’s t test (B, F, H, I, and J) and two-way ANOVA for multiple comparisons (G), *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, and ****P ≤ 0.0001. # above pooled data in violin plots (right, G) indicates empirical P values calculated by the Nested Leave-One-Out Jackknife strategy, where #P < 0.5–0.01, and ###P < 0.001.