Figure 1.
Figure 1. PC degranulation in intestinal epithelial organoids after muscarinergic but not after bacterial stimulation. (A) Small intestinal organoids were stimulated with 10 µg/ml PGN, 100 µg/ml poly(I:C), 1 µg/ml LPS, 0.1 µg/ml flagellin, 1 µg/ml CpG ODN, 100 µg/ml MDP, heat-inactivated (h.i.), or live E. coli bacteria or Cch (25 µM) for 12 h, and granules were visualized by UEA-1 lectin staining. Nonstimulated cultures served as controls. PC granules are indicated by blue arrowheads; granule release into the crypt lumen is indicated by the blue arrow. Whole organoids (top) and magnified crypts (bottom) are shown. Bars, 100 µm. (B) Intact organoids in Matrigel (top) were stimulated basolaterally (12 h) or organoid fragments (bottom) were stimulated both apically and basolaterally (2 h), with the indicated bacterial patterns and lysozyme expression assessed by Western blot. Cells were washed to remove luminal lysozyme. α-Tubulin was probed for normalization. Experiments were performed in three parallel cultures. (C) Quantification of lysozyme content from Western Blot data in B. Densitometric analysis of lysozyme signals normalized to α-tubulin expression. Error bars show standard deviation; **, P < 0.01; Student’s t test. Results in A–C were reproduced in two independent experiments. (D) Expression of the indicated microbial pattern receptors; RT-PCR analysis using cDNA from isolated crypts and cultured organoids. Hprt was analyzed for normalization. Representative data of three independent experiments. (E) Intact organoids and organoid fragments were stimulated with the indicated stimuli for 1 h, and Icam1 expression was assessed by qPCR analysis. Mean relative expression ± SD in n = 3 parallel wells; **, P < 0.01; ***, P < 0.001; Student’s t test. Data representative of three independent experiments. (F) Human colon cancer cell lines Caco-2 and HT-29 were stimulated with the indicated stimuli for 1 h, and IL-8 expression was assessed by qPCR. Mean relative expression ± SD (log10 scale) in n = 3 parallel wells; *, P < 0.05; **, P < 0.01; ***, P < 0.001; Student’s t test. Experiment has been performed twice.

PC degranulation in intestinal epithelial organoids after muscarinergic but not after bacterial stimulation. (A) Small intestinal organoids were stimulated with 10 µg/ml PGN, 100 µg/ml poly(I:C), 1 µg/ml LPS, 0.1 µg/ml flagellin, 1 µg/ml CpG ODN, 100 µg/ml MDP, heat-inactivated (h.i.), or live E. coli bacteria or Cch (25 µM) for 12 h, and granules were visualized by UEA-1 lectin staining. Nonstimulated cultures served as controls. PC granules are indicated by blue arrowheads; granule release into the crypt lumen is indicated by the blue arrow. Whole organoids (top) and magnified crypts (bottom) are shown. Bars, 100 µm. (B) Intact organoids in Matrigel (top) were stimulated basolaterally (12 h) or organoid fragments (bottom) were stimulated both apically and basolaterally (2 h), with the indicated bacterial patterns and lysozyme expression assessed by Western blot. Cells were washed to remove luminal lysozyme. α-Tubulin was probed for normalization. Experiments were performed in three parallel cultures. (C) Quantification of lysozyme content from Western Blot data in B. Densitometric analysis of lysozyme signals normalized to α-tubulin expression. Error bars show standard deviation; **, P < 0.01; Student’s t test. Results in A–C were reproduced in two independent experiments. (D) Expression of the indicated microbial pattern receptors; RT-PCR analysis using cDNA from isolated crypts and cultured organoids. Hprt was analyzed for normalization. Representative data of three independent experiments. (E) Intact organoids and organoid fragments were stimulated with the indicated stimuli for 1 h, and Icam1 expression was assessed by qPCR analysis. Mean relative expression ± SD in n = 3 parallel wells; **, P < 0.01; ***, P < 0.001; Student’s t test. Data representative of three independent experiments. (F) Human colon cancer cell lines Caco-2 and HT-29 were stimulated with the indicated stimuli for 1 h, and IL-8 expression was assessed by qPCR. Mean relative expression ± SD (log10 scale) in n = 3 parallel wells; *, P < 0.05; **, P < 0.01; ***, P < 0.001; Student’s t test. Experiment has been performed twice.

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