Fig 1: IL-11 induces signals to colonic epithelial cells and fibroblasts.a Colonic epithelial organoids and fibroblasts were established from three independent wild-type mice as described in the methods. Expressions of the indicated genes were determined using qPCR. Results are mean ± SEM (n = 3 mice). Results are representative of two independent experiments. b Colonic epithelial organoids and fibroblasts were unstimulated or stimulated for 30 min with IL-11 (10 or 100 ng/mL) or IL-22 (10 or 100 ng/mL). Total STAT3 and phosphorylated STAT3 (pSTAT3) were analyzed by Western blotting. STAT3 and pSTAT3 signaling intensities were calculated by Fiji, and the relative ratio of pSTAT3/STAT3 is shown. Results are representative of two independent experiments (a, b). c, d Relative expression of Il11ra1 and Il6st in colon organoids. Colon organoids were established from WT and Apcdelta716 mice carrying a truncated Apc gene. Expression of the indicated genes in WT and Apcdelta716 organoids was determined using qPCR (c). Results are mean ± SEM (n = 3 independently established organoids from different mice). Colon organoids were not stimulated or stimulated with IL-11 (100 ng/mL) for 30 min (d). Total STAT3, phosphorylated STAT3 (pSTAT3), total ERK, and phosphorylated ERK (pERK) were analyzed and described as in (b). Results are representative of two independent experiments. e, f Administration of IL-11R agonist induces expression of the genes expressed in IL-11+ cells. We injected 8-week-old wild-type mice with 10 µg IL-11R agonist. At 3 h after injection, mRNA was isolated from the whole colon, and gene expression was analyzed using microarray analysis as described in “Methods” (n = 2 for untreated samples; n = 3 for injected samples). Volcano plot of whole genes (e). The horizontal line indicates differentially regulated genes in the whole colon after IL-11R agonist injection compared with the whole colon before injection, shown in log2. The vertical line indicates P values, shown in -log10. Red dots indicate significantly upregulated genes. Venn diagram of genes enriched in FACS-sorted IL-11+ fibroblasts and genes with elevated expression in the whole colon after IL-11R agonist treatment compared with an untreated colon (f). Nineteen overlapping genes are shown. g Venn diagram of genes enriched in FACS-sorted IL-11+ fibroblasts and genes induced by IL-11 stimulation in ColVI+ fibroblasts. Five overlapping genes are shown. Statistical significance was determined using the two-tailed unpaired Student’s t-test (a, c). p-values were calculated by two-tailed local-pooled-error test (e). Source data are provided as a Source Data file.
Fig 2: Both IL-17 and IL-22 induce an increase in IKKα expression in Neoderm. Neoderm tissues were cultured under 100 ng/ml of IL-17 or IL-22 for 48 hours and fixed for H&E and immunohistochemical staining assays. The results shown are representative of the three independent batches of Neoderm experiments.
Fig 3: Cell cycle analysis demonstrates that HaCaT cells cultured by switching into high calcium concentrations (Ca 2.8 mM) evidenced an elevated proportion of G0/G1 phase, regardless of IL-17 and IL-22 treatment. By way of contrast, HaCaT cells maintained at low calcium concentrations (Ca 0.06 mM) evidenced more cells in S- or G2/M phase compared with those transferred to a medium containing 2.8 mM of calcium. The data are expressed as the means±standard error of the mean and the statistical differences were analyzed between low- and high-calcium treated groups in each of the experiments. PI=(S+G2/M)/(G0/G1+S+G2/M). NS: not significant, PI: proliferation index. **p<0.01; *p<0.05.
Fig 4: IL-17 and IL-22 induce pro-inflammatory cytokine production in HaCaT cells. HaCaT cells were cultured under 100 ng/ml of IL-17 or IL-22 for 48 hours, with or without preincubation of PS1145, at a concentration of 10µM for an hour and collected culture supernatant for ELISA. The data are expressed as the means±standard error of the mean at three independent experiments. NS: not significant. *p<0.05.
Fig 5: Elevated IKKα levels in inflammatory mice ears, evoked by IL-17 and IL-22 injection. Skin inflammation, edema, and acanthosis, which were involved in the infiltration of neutrophils and dendritic cells into the ear skin of C57BL/6 WT mice, but less profound inflammatory changes, were noted in the sham controls. Each ear of C57BL/6 WT mice were injected intradermally with 3µg of IL-17 and IL-22 for two consecutive days in a total volume of 30µl. The control group of mice was injected with the same volumes of phosphate-buffered saline (PBS) via an identical route.
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