Enteric neuron-intrinsic HIF1α signaling limits CCL2-driven monocyte recruitment and protects from myenteric plexus remodeling and postinflammatory GI dysmotility. (A) Confocal images showing cross-sections of colons isolated from 1×DSS- (3.5%) or water-treated and EF5- or vehicle-injected WT mice at week 1, stained with EF5 (hypoxic cells) and Hu (neuronal somas) antibodies, and DAPI (nuclei). Scale bars, 50 μm. (B) Percentage of total EF5⁺ Hu⁺ hypoxic neurons in cross-sections of colons of mice described in A (n = 2 per group, n—cross-section of full-length colon Swiss roll). (C) Graphical summary of HIF signaling pathway. (D)Hif1a expression by qPCR in colonic myenteric neurons FACS-purified from 1×DSS- or water-treated mice at week 1, normalized to Actb (n = 4–5 per group, n—independent cell sorting). (E)Vegfa expression by qPCR in colonic myenteric neurons FACS-purified from 1×DSS- or water-treated mice at week 1, normalized to Actb. (n = 6 per group, n—independent cell sorting). (F)Ccl2 expression by qPCR in cultured adult colonic myenteric neurons treated with CoCl₂, LPS, or their combinations, normalized to Actb (n = 3 per group, n—replica wells in the same cell culture). (G) CCL2 concentration by ELISA in supernatants from cultured adult colonic myenteric neurons described in F (n = 3 per group, n—replica wells in the same cell culture). (H)Ccl2 expression by qPCR in cultured adult colonic myenteric neurons treated with the HIF1α inhibitor PX-478 or vehicle, normalized to Actb (n = 3 per group, n—replica wells in the same cell culture). (I) Experimental groups used in J and K, analyzed at week 1 after starting DSS. (J)Ccl2 expression by qPCR in CD45^–CD90⁺CD24⁺ colonic myenteric neurons FACS-purified from 1×DSS (2.5%)- or water-treated WT mice daily injected with PX-478 or vehicle, analyzed at week 1, and normalized to Actb (n = 6 per group). (K) Percentage of total viable cells (left) and absolute cell counts (right) of colonic Mo-MMs, quantified by FC (DSS vehicle and PX-478, n = 8 per group). Baseline shows the mean value for PX-478–injected water-treated mice (n = 1). (L) Experimental groups used in M–P, analyzed at weeks 1 and 11 after starting DSS. (M) Percentage of total viable cells (left) and absolute cell counts (right) of colonic Mo-MMs from tamoxifen- and 1× or 3×DSS-treated (2.5%) SLICK-H^ER-CreVhl^fl/fl (Cre⁺) and Vhl^fl/fl littermates (Cre^–), quantified by FC (DSS Cre^– and Cre⁺, n = 7–19 per group). Baseline shows the mean value for tamoxifen- and water-treated Vhl^fl/fl littermates (Water Cre^–, n = 5–9). P values above each column indicate statistical differences against baseline. (N) Structural changes in colonic myenteric plexus of mice described in M, quantified by confocal microscopy, showing counts of neuron clusters, counts of Hu⁺ neurons per cluster, and area and counts of IGFT regions. Data are shown as distributions of pooled values generated from analysis of 5–8 fields of view per mouse per group (DSS Cre^– and Cre⁺, n = 4–7 per group; water Cre^–, n = 2–3). (O) Contacts between MHCII⁺ MMs and Hu⁺ myenteric neurons of mice described in M, analyzed at week 1 (DSS Cre^– and Cre⁺, n = 4 per group). Baseline shows the mean value for tamoxifen- and water-treated Vhl^fl/fl littermates (water Cre^–, n = 2). Data are shown as distributions of pooled values generated from analysis of 5 fields of view per mouse per group. (P) GI transit of mice described in M, analyzed at week 11 (DSS Cre^– and Cre⁺, n = 7 per group). Baseline shows the mean value for tamoxifen- and water-treated Vhl^fl/fl littermates (water Cre^–, n = 5). P values above each column indicate statistical differences against baseline. All graphs show the mean ± SEM, n—mouse, unless stated differently. Data are representative of at least two independent experiments with similar results. Data are pooled from two to four smaller experiments (D, E, K, and M). Statistical analyses: unpaired Student’s t test (D–H, J, K, M, O, and P) and two-way ANOVA for multiple comparisons (N), *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, and ****P ≤ 0.0001. # above pooled data in violin plots (N) indicates empirical P values calculated by the Nested Leave-One-Out Jackknife strategy, where #P < 0.05, ##P < 0.01, and ###P < 0.001.