TLR4⁺ immune cells induce epithelial NF-κB activation in the small intestine and colon. (A) Schematic drawing of the two-photon imaging (left). The intestinal mucosa is imaged from the luminal side (black arrowhead), resulting in images in horizontal plane of the mucosa (right; part of the image shown in Fig. 1 A). White dashed line/E, epithelium; L, lumen; white asterisks, epithelial nuclei. (B) Fold changes in expression of A20, Cxcl2, and Tnf in the cecal mucosa of mice depicted in Fig. 1 A (n = 5). (C) Fold changes in expression of A20, Cxcl2, and Tnf in the cecal mucosa of mice depicted in Fig. 1 B in comparison to PBS-injected mice in Fig. 1 A (n = 5). (D and E) Two-photon microscopy images and quantification of epithelial NF-κB activation in the (D; n = 3–5) small intestine and (E; n = 3–5) colon of LPS-injected BMCs, and (F) in small intestine and colon of Myd88^−/− > p65^GFP-FLxTlr4^−/−, Ticam1^−/− > p65^GFP-FLxTlr4^−/− BMCs. Each circle represents one mouse. Black line: median. Statistical analysis: Mann–Whitney U test. *, P ≤ 0.05; **, ≤ 0.01. Scale bars: 50 µm. Combined data of three (B), four (C), five (D), six (E), or seven (F) independent experiments. Each circle represents one mouse. Black line: median. Scale bars: 50 µm.