Figure 5.
TNF-mediated epithelial NF-κB activation occurs upon bacterial infection and induces an antibacterial response. (A) Representative two-photon microscopy images of cecal explants of streptomycin-pretreated p65GFP-FL mice infected with S. Tm for 8–13.5 h (n = 28). Categories for scoring of epithelial NF-κB activation status: “no activation” (green); “patchy activation” (blue); “full activation” (orange); “inflammation” (red; tissue distortion evident); “unspecified” (gray; was excluded from further analysis). Scale bars: 50 µm. (B) Distribution of the analyzed 28 samples of A among the four epithelial NF-κB activation categories (bottom). For simplification, the blue, orange, and red categories were summarized as “NF-κB signaling” (yellow, top). (C)Tnf transcript levels in the cecal mucosa of mice described in A and naive p65GFP-FL mice, grouped according to the epithelial NF-κB activation status of the respective mice and depicted as 2-ΔCT. Expression levels were normalized to Actb (n = 33). (D)TNFa+/− or TNFa−/− > p65GFP-FLxTlr4−/− BMCs were analyzed as described in A and B. (E) Log2 ratios of selected genes in a transcriptome analysis of TNF-treated (8 h, 5 ng/ml) compared with untreated small intestinal epithelial organoids (Hausmann et al., 2020b). FDR, false discovery rate. (F)C3 transcript levels in untreated and TNF-treated (5 ng/ml, 4 h) small intestinal organoids depicted as 2-ΔCT. Expression levels were normalized to Actb. (G) Streptomycin-pretreated TNFa−/− mice and heterozygous littermates were orally infected with S. Tm for 36h. S. Tm in the cecal lumen (gated on O12+ cells, see Fig. S5 K) were stained for surface C3 to assess coating of luminal bacteria by flow cytometry (C3+ population). MFI, median fluorescence intensity. Statistical analysis: Mann–Whitney U test (C, F, and G) or χ2 test (D). *, P ≤ 0.05; **, P ≤ 0.01. Each circle represents one mouse (C and G) or one experiment (average; F). y axis in log10 scale (C, F, and G). Combined data of three (G), four (F), five (A–C), or six (D) independent experiments.

TNF-mediated epithelial NF-κB activation occurs upon bacterial infection and induces an antibacterial response. (A) Representative two-photon microscopy images of cecal explants of streptomycin-pretreated p65GFP-FL mice infected with S. Tm for 8–13.5 h (n = 28). Categories for scoring of epithelial NF-κB activation status: “no activation” (green); “patchy activation” (blue); “full activation” (orange); “inflammation” (red; tissue distortion evident); “unspecified” (gray; was excluded from further analysis). Scale bars: 50 µm. (B) Distribution of the analyzed 28 samples of A among the four epithelial NF-κB activation categories (bottom). For simplification, the blue, orange, and red categories were summarized as “NF-κB signaling” (yellow, top). (C)Tnf transcript levels in the cecal mucosa of mice described in A and naive p65GFP-FL mice, grouped according to the epithelial NF-κB activation status of the respective mice and depicted as 2-ΔCT. Expression levels were normalized to Actb (n = 33). (D)TNFa+/− or TNFa−/− > p65GFP-FLxTlr4−/− BMCs were analyzed as described in A and B. (E) Log2 ratios of selected genes in a transcriptome analysis of TNF-treated (8 h, 5 ng/ml) compared with untreated small intestinal epithelial organoids (Hausmann et al., 2020b). FDR, false discovery rate. (F)C3 transcript levels in untreated and TNF-treated (5 ng/ml, 4 h) small intestinal organoids depicted as 2-ΔCT. Expression levels were normalized to Actb. (G) Streptomycin-pretreated TNFa−/− mice and heterozygous littermates were orally infected with S. Tm for 36h. S. Tm in the cecal lumen (gated on O12+ cells, see Fig. S5 K) were stained for surface C3 to assess coating of luminal bacteria by flow cytometry (C3+ population). MFI, median fluorescence intensity. Statistical analysis: Mann–Whitney U test (C, F, and G) or χ2 test (D). *, P ≤ 0.05; **, P ≤ 0.01. Each circle represents one mouse (C and G) or one experiment (average; F). y axis in log10 scale (C, F, and G). Combined data of three (G), four (F), five (A–C), or six (D) independent experiments.

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