Fig. 4. Pronounced NLRC4-independent dislodging of enterocytes at 24 h p.i. involves TNF.

a, b NLRC4 deficiency leads to pronounced epithelial erosion. a Representative micrograph of cecal tissue from S.Tm/pssaG-GFP-infected Nlrc4^−/− mice at 18 h p.i. This pronounced erosion can only be seen occasionally in a few mice at this time point. Arrows indicate epithelial S.Tm-G⁺. Arrowheads indicate dislodged enterocytes with and without intracellular S.Tm-G⁺. Lu. lumen. Scale bar: 50 µm. b Microscopy-based quantification of enterocyte fraction among dislodging cells in a. Line at median. c, d Microscopy-based characterization of dislodging enterocytes. c Representative micrographs of cecal tissue from Nlrc4^−/− mice at 18 h p.i. stained for ASC, cleaved Caspase-3 and cleaved Caspase-8. Lu. lumen. Scale bar: 20 µm. d Microscopy-based quantification of the indicated markers and fragmented nuclei in dislodging enterocytes. Each data point represents one field of view. Four mice quantified. Line at median. e–g S.Tm/pssaG-GFP-infection of Nlrc4^−/−Nlrp3^+/− control mice and Nlrc4^−/−Nlrp3^−/− littermates for 24 h. e Representative micrographs of cecal tissue. F Microscopy-based quantification of dislodged enterocytes per 40× field of view. g Microscopy-based quantification of intracellular S.Tm-G⁺ in cecal epithelium. h–j S.Tm/pssaG-GFP infection of Nlrc4^−/−Caspase11^+/− control mice and Nlrc4^−/−Caspase11^−/− littermates for 24 h. h Representative micrographs of cecal tissue. I Microscopy-based quantification of dislodged enterocytes per 40× field of view. j Microscopy-based quantification of intracellular S.Tm-G⁺ in cecal epithelium. k–m S.Tm/pssaG-GFP infection of Nlrc4^−/− mice for 24 h, injected (i.p.) with anti-IFNγ or isotype control antibody (twice daily dose of 500 µg antibodies per mouse, starting 1 day before infection¹⁰³). k Representative micrographs of cecal tissue. l Microscopy-based quantification of dislodged enterocytes per 40× field of view. m Microscopy-based quantification of intracellular S.Tm-G⁺ in cecal epithelium. n–p S.Tm/pssaG-GFP infection of Nlrc4^−/− mice for 24 h, injected (i.p.) with anti-TNF or isotype control antibody (once with 500 µg antibodies per mouse 1 day before infection). n Representative micrographs of cecal tissue. o Microscopy-based quantification of dislodged enterocytes per 40× field of view. p Microscopy-based quantification of intracellular S.Tm-G⁺ in cecal epithelium. e, h, k, n Lu.—lumen, scale bars: 50 µm. f, g, i, j, l, m, o, p Each data point represents one mouse. Line at median. Five to seven mice per group from ≥2 independent experiments for each comparison. Note: Expression and maturation of GFP reporter takes around 2–4 h. Mann–Whitney U test (ns—not significant, **p < 0.01).