Fig 1: The IL-33-induced IA decrease requires Syk. A, effects of 50 ng/mL IL-33 on the protein abundance of phospho-JAK2 (p-JAK2) or total JAK2 (t-JAK2). Blots are representative of three independent experiments with β-tubulin serving as a loading control. B, colabeling (white arrows) of ST2 and JAK2 and Syk in mouse DRG sections. Scale bar, 50 µm. C, time course of IA changes (left) and bar graph (right) showing that pretreatment of DRG neurons with AG490 (10 µM) did not affect the IL-33-induced IA response (n = 8 cells). Application of 10 µM AG490 alone did not affect IA (n = 7 cells). Arabic numerals indicate the points utilized for the example current traces. D, effects of 50 ng/mL IL-33 on p-Syk protein abundance in the presence or absence of the ST2 neutralizing antibody (ST2 Ab, 2 µg/mL) in DRG cells. Blots are representative of three independent experiments with β-tubulin serving as a loading control. **p < 0.01 vs. control, unpaired t test. E-G, time course of the IA changes showing that pretreating DRG neurons with R406 or GS9973, but not KT-5720, prevented the IL-33-induced IA response. Arabic numerals indicate the points utilized for the example current traces. H, bar graph showing the effect of 50 ng/mL IL-33 on IA in cells pretreated with R406 (1 µM, n = 11 cells), GS9973 (10 µM, n = 8 cells), and KT-5720 (1 µM, n = 9 cells). Application of 1 µM R406 (n = 7 cells), 10 µM GS9973 (n = 6 cells) or 1 µM KT-5720 (n = 7 cells) alone had no significant effect on IA. **p < 0.01 vs. control, paired t test. I, colabeling (white arrows) of ST2 and PKA in mouse DRG sections. Scale bar, 50 µm. J, bar graph demonstrating the effect of 20 µM forskolin on IA in the presence (n = 7 cells) or absence (n = 5 cells) of KT-5720 (1 µM). ***p < 0.001 vs. forskolin without KT-5720, paired t test.
Fig 2: IL-33 negatively regulates epithelial barrier function in the colon. (A) Disease activity index of 3% dextran sodium sulphate-treated mice that received intraperitoneal injections of 0.5 µg recombinant IL-33 on days 1, 3 and 5 is depicted. Mean values±SEM are given (n=4 per group). (B) ST2 expression on Caco-2 cells. (C) Transepithelial electrical resistance of Caco-2 monolayers treated with indicated doses of recombinant IL-33 at the indicated time points. Pooled data of three independent experiments are shown, bars represent means±SD. (D) Intestinal permeability in placebo and rmIL-33-treated mice assessed by measuring serum FITC-Dextran levels 4 h after administration. Data are displayed as mean±SEM with n=4 per group. One representative of two independent experiments is shown. *p<0.05, unpaired t test. (E) Colonic expression of endogenousCx43 and IL-33 is shown in (E) untreated and (F) IL-33-treated mice.
Fig 3: IL-33 and St2 expression are induced in CRC in mice. (A) IHC for IL-33 of healthy and tumor WT intestinal tissue. Scale bars: overview: 200 µm; inlay 25 µm. (B) Increased St2 transcript levels in WT tumor vs. adjacent tumor-free tissue. Data represent means ± SEM; n = 9 samples per group; n.d., non-detected. Statistical analysis was performed using paired t test.
Fig 4: IL-33/ST2 is co-expressed in trophoblasts. Immunohistochemical analysis (n=10) for IL-33 and ST2 expression in villi tissues from healthy women at the first-trimester of a normal pregnancy. Magnification, ×200 or ×400. IL-33, interleukin 33; ST2, IL-1 receptor-like 1.
Fig 5: ST2 in human inflammatory bowel diseases and in experimental models of colitis. (A) Relative mouse ST2 mRNA expression was determined by qRT-PCR analysis for different cells types and normalised using Actb (n=4). (B) Immunohistochemical analysis of human ST2 expression in controls, active Crohn's disease, active ulcerative colitis (UC) and UC in remission patients. Size bar, 100 µm. (C) Immunohistochemical analysis of mouse ST2 expression on day 5 and on day 8 of DSS-induced colitis. Inserts represent enlargements of the boxed areas. Lower edges of the large panels: 689 µm length, of the inserts: 143 µm. Data show means±SEM. A second independent experiment gave similar results.
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