Figure S3.
The influence of age and MyD88 signaling for enterocyte efferocytosis in the stem cell organoid co-culture model. (A) Comparative quantitative analysis of the number of internalization events of tdTomato-positive growth factor–starved cells/cell material (starved IECs) derived from adult stem cell organoids (starved adult IECs) or neonatal stem cell organoids (starved neonatal IECs) by intestinal epithelial stem cell organoid–derived cell monolayers generated from neonatal (N) or adult (AD) small intestines per mm2. 30–49 images (1.25 × 1.00 mm) obtained from two to three independent experiments were analyzed. The data for cell monolayers exposed to starved adult IECs are identical to Fig. 2 D. One-way ANOVA Kruskal–Wallis test with Dunn’s post-test. ****, P < 0.0001; ns, non-significant. (B) TNF-α (pg/ml) in the cell culture supernatant of adult intestinal epithelial stem cell organoid–derived cell monolayers left untreated, stimulated with S. Typhimurium (MOI 10:1), or exposed to growth factor–starved intestinal epithelial stem cell organoid cells (starved IECs) for 2 h. Two independent experiments with three replicates were analyzed; the graph shows one representative experiment. One-way ANOVA Kruskal–Wallis test with Dunn’s post-test. **, P < 0.01; ns, non-significant. (C) Immunostaining for CD36 (orange), cleaved caspase 3 (cl. Casp3, red) and S. Typhimurium (green) in small intestinal tissue sections of healthy 5-day-old mice. Counterstaining with DAPI (blue). Bar, 20 μm. A representative image is shown. (D) TEM images of an S. Typhimurium–infected IEC with signs of reduced viability. The white squares indicate areas that are displayed at higher resolution in adjacent panels on the right. Microvilli at the cell surface are vesiculated, and the cell cytoplasm appears vacuolized and extracted. The electron-dense, amorphous material represents the luminal contents of the SCV-like endosome. Bacteria are visible inside the SCV in the left cell. S, S. Typhimurium. Bar, 5 µm (left and middle panel), 1 µm (right panel). (E–G) 1-day-old MyD88+/+ and MyD88−/− mice were orally infected with 102 CFU S. Typhimurium (S. Tm). Small intestinal tissue sections at day 4 p.i. were used to quantify the percentage of (E) S. Typhimurium–positive cells among all efferosome-positive IECs and (F) S. Typhimurium–positive efferosome positive cells among all S. Typhimurium–positive IECs. 18–27 image fields with the size of 312.35 × 250.38 µm of small intestinal tissue sections obtained at day 4 p.i. from three to five individual S. Typhimurium–infected neonatal animals were analyzed. Mann–Whitney U test. **, P < 0.01; ****, P < 0.0001. (G)Salmonella organ load in isolated gentamicin-treated enterocytes from MyD88+/+ and MyD88−/− mice at day 4 p.i. (n = 2–4).

The influence of age and MyD88 signaling for enterocyte efferocytosis in the stem cell organoid co-culture model. (A) Comparative quantitative analysis of the number of internalization events of tdTomato-positive growth factor–starved cells/cell material (starved IECs) derived from adult stem cell organoids (starved adult IECs) or neonatal stem cell organoids (starved neonatal IECs) by intestinal epithelial stem cell organoid–derived cell monolayers generated from neonatal (N) or adult (AD) small intestines per mm2. 30–49 images (1.25 × 1.00 mm) obtained from two to three independent experiments were analyzed. The data for cell monolayers exposed to starved adult IECs are identical to Fig. 2 D. One-way ANOVA Kruskal–Wallis test with Dunn’s post-test. ****, P < 0.0001; ns, non-significant. (B) TNF-α (pg/ml) in the cell culture supernatant of adult intestinal epithelial stem cell organoid–derived cell monolayers left untreated, stimulated with S. Typhimurium (MOI 10:1), or exposed to growth factor–starved intestinal epithelial stem cell organoid cells (starved IECs) for 2 h. Two independent experiments with three replicates were analyzed; the graph shows one representative experiment. One-way ANOVA Kruskal–Wallis test with Dunn’s post-test. **, P < 0.01; ns, non-significant. (C) Immunostaining for CD36 (orange), cleaved caspase 3 (cl. Casp3, red) and S. Typhimurium (green) in small intestinal tissue sections of healthy 5-day-old mice. Counterstaining with DAPI (blue). Bar, 20 μm. A representative image is shown. (D) TEM images of an S. Typhimurium–infected IEC with signs of reduced viability. The white squares indicate areas that are displayed at higher resolution in adjacent panels on the right. Microvilli at the cell surface are vesiculated, and the cell cytoplasm appears vacuolized and extracted. The electron-dense, amorphous material represents the luminal contents of the SCV-like endosome. Bacteria are visible inside the SCV in the left cell. S, S. Typhimurium. Bar, 5 µm (left and middle panel), 1 µm (right panel). (E–G) 1-day-old MyD88+/+ and MyD88−/− mice were orally infected with 102 CFU S. Typhimurium (S. Tm). Small intestinal tissue sections at day 4 p.i. were used to quantify the percentage of (E) S. Typhimurium–positive cells among all efferosome-positive IECs and (F) S. Typhimurium–positive efferosome positive cells among all S. Typhimurium–positive IECs. 18–27 image fields with the size of 312.35 × 250.38 µm of small intestinal tissue sections obtained at day 4 p.i. from three to five individual S. Typhimurium–infected neonatal animals were analyzed. Mann–Whitney U test. **, P < 0.01; ****, P < 0.0001. (G)Salmonella organ load in isolated gentamicin-treated enterocytes from MyD88+/+ and MyD88−/− mice at day 4 p.i. (n = 2–4).

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