TNF produced by CD11c⁺ cells induces local epithelial NF-κB activation in the intestinal mucosa. (A and B) p65^GFP-FL intestinal epithelial organoids established from the indicated regions were treated with 5, 50, and 500 ng/ml or 5 µg/ml LPS (+ LBP and CD14, if indicated) and imaged for 1 h (A; n = 3–17), or analyzed by qPCR at 3 h of treatment (B and C; n = 6 or 7). (C) Colon organoids from p65^GFP-FLxTlr4^−/− mice (n = 4). (D) Representative two-photon microscopy overview image of the cecal mucosa of mice described in Fig. 2 A at 1 h.p.inj. of LPS (n = 6). Red squares indicate RFP⁺ (Tlr4^+/+) cells. White lines indicate IEC NF-κB activation zones (defined as areas with continuous epithelial NF-κB activation). (E) Quantification of epithelial NF-κB activation in Il18^−/− > p65^GFP-FLxTlr4^−/−, Il18r^−/− > p65^GFP-FLxTlr4^−/−, and Il1ab^−/− > p65^GFP-FLxTlr4^−/− BMCs at 1 h.p.inj. of LPS (n = 7 or 8). (F) p65^GFP-FL intestinal epithelial organoids from cecum (left) or colon (right) were treated with 5, 50, and 500 ng/ml TNF and imaged for 1 h (n = 3–17). (G) Quantification of epithelial NF-κB activation in mice as described in Fig. 2 B. Mice pretreated with DTX were injected with PBS or TNF (n = 2–6). Cecae were imaged at 1 h.p.inj. Data of LPS-injected mice are replotted from Fig. 2 B for comparison. Black line: median (B, C, E, and G). Dashed line: detection limit (C and G) or error range (A and F). Each circle represents one organoid sample (B and C), one mouse (E and G), or the median (A and F). Statistical analysis: one-way ANOVA with Dunett’s correction (B and C) or Mann–Whitney U test (E and G). *, P ≤ 0.05; **, P ≤ 0.01. Scale bars: 50 µm. Combined data of two (A, small intestine; B, C, D, and F, cecum), three (A, cecum), four (F, colon), six (B and E), or eight (A, colon) independent experiments.