Tissue resident, monocyte-derived macrophages secrete TNF to induce local epithelial NF-κB activation. (A) ELISA measurements of TNF concentrations in the cecal mucosa of LPS injected WT mice (n = 5 or 6). Dashed line: detection limit. y axis in log₁₀ scale. (B) Percentage of TNF⁺ DCs or macrophages (gating as shown in Fig. S4 D) in the cecum, small intestine, and colon of LPS-treated WT mice (1 h.p.inj.) and PBS-treated controls (n = 5–7). (C) Representative images of the cecal epithelium and quantification of epithelial NF-κB activation of p65^GFP-FL mice pretreated with anti-CSF1R or isotype control, injected with LPS, and imaged 1 h.p.inj. (n = 7). Depletion efficiency of macrophages and DCs in anti-CSF1R treated mice. (D) Normalized marker expression of TNF⁺ compared with TNF⁻ macrophages in the cecum, small intestine, and colon of LPS-injected WT mice (n = 4–7). (E) Percentage of CD4^+/− Tim4^+/− cells among TNF⁻ and TNF⁺ macrophages in the cecum of LPS-injected WT mice (n = 4). (F) TNF-PLA analysis of cecae from p65^GFP-FLxTlr4^−/− mice reconstituted with a 1:40 mix of ActRFP (2.5%, Tlr4^+/+) and p65^GFP-FLxTlr4^−/− (97.5%) BM. Representative confocal microscopy image of fixed cecal tissue at 40 min.p.inj. (left) and quantification of PLA for TNF in crypts without (−) or with (+) epithelial NF-κB activation (Fig. S5 A) at 1 h.p.inj. (n = 11–13). Scale bar: 10 µm. Black line: median. Statistical analysis: one-way ANOVA with Dunett’s correction (A), two-way ANOVA with Sidak’s multiple comparison test (B), or Mann–Whitney U test (C, E, and F). *, P ≤ 0.05; **, P ≤ 0.01. Each circle represents one mouse (A–E) or one crypt (F; five mice analyzed). Combined data of two (D), three (B and C), four (F), or six (A) independent experiments, or exemplary data of two (E) independent experiments.