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hep26446-sup-0001-suppfig1.tif334KFigure S1. A-B. Hepatocytes and Mononuclear cells were isolated from the liver of 4 normal mice and analyzed for the expression of albumin (A) and CD3 (B) by Real-Time PCR.
hep26446-sup-0002-suppfig2.tif433KFigure S2. A. Mice, injected intra-peritoneally (ip) with D-Galactosamine (D-Gal) and lipopolysaccharide (LPS), were sacrificed 8 hours later and livers were explanted and used for RNA extraction. RNA transcripts of Fizz were measured by real-time PCR. Data show the mean ± SEM of all samples (N=9 mice per group). B. Mice, injected intra-peritoneally (ip) with D-Galactosamine (D-Gal) and lipopolysaccharide (LPS), were sacrificed at the indicated time-points and livers were explanted and used for RNA extraction. RNA transcripts of α-fetoprotein were measured by real-time PCR. Data show the mean ± SEM of all samples (N=10 mice per each time point). C. IL-25 protein expression was measured in the supernatants of liver explants taken from BALB/c mice and cultured in the presence of PBS or D-Gal (100µM) and LPS (500ng/ml) for 24 hours. Data are the mean ± SD of three independent experiments; *p<0.05.
hep26446-sup-0003-suppfig3.tif559KFigure S3: Representative dot plots showing the percentage of AML12 cells expressing AnnexinV (AV) and/or propidium iodide (PI). Cells were pre-treated with Actinomycin D (250 ng/ml) or DMSO (vehicle, diluted 1:100 in PBS) for 30 minutes and then cultured in the presence or absence of IL-25 (50ng/ml) and/or TNF-α (25ng/ml). One of 3 independent experiments in which similar results were obtained is shown.
hep26446-sup-0004-suppfig4.tif1384KFigure S4. IL-25 reduces D-Gal/LPS-induced IL-23/p19 expression in mice with D-GAL/LPS-induced fulminant hepatitis (FH). Mice were given intraperitoneally IL-25 or vehicle 1 hour before treatment with D-Gal/LPS or PBS. Mice were sacrificed 8 hours after D-Gal/LPS treatment and livers were explanted and used to measure the RNA expression of IL-23p19 (A), IL-17A (B) and IL-22 (C) by real-time PCR. Histograms show the mean ± SEM of all samples (N= at least 6 mice per group); *p<0.05.
hep26446-sup-0005-suppfig5.tif2556KFigure S5. A-D. IL-25 enhances the expression of MDSC-associated immunesuppressive molecules in D-Gal/LPS-injured mice. Mice were given intraperitoneally IL-25 or vehicle 1 hour before treatment with D-Gal/LPS or PBS and were sacrificed 8 hours after D-Gal/LPS treatment. Livers were explanted and used to measure the RNA expression of arginase 2 (Arg2; A) and iNOS (B) by real-time PCR. Histograms show the mean ± SEM of all samples (N= 6 mice per group); *p<0.05; **p<0.01. C-D. MDSC (GR1/CD11b+ cells) isolated from the liver of D-Gal/LPS-treated mice receiving IL-25 express high levels of Arg2 (C) and iNOS (D). Histograms show the data generated using GR1/CD11b- and GR1/CD11b+ cells sorted from the liver cell preparations of 6 mice per group, pooled and analyzed by real-time PCR. E-F. Adoptive transfer of MDSC protect mice from D-Gal/LPS-induced FH. MDSC purified from the spleen of mice, were resuspended in PBS, and adoptive transferred in recipient mice trough the caudal vein. All mice were given D-Gal/LPS 1 hour after cell transfer (N=6 mice per group). Control mice received PBS alone. E. Serum levels of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) were measured 6 hours after Gal/LPS-treatment. Data indicate the mean ± SEM of all mice; *p<0.05; **p<0.001; F. Representative photomicrographs of haematoxylin & eosin-stained liver sections of mice treated as in E and euthanized 8 hours after Gal/LPS treatment (original magnification 100x).
hep26446-sup-0006-suppfig6.tif275KFigure S6. GR1/CD11b-positive cells isolated from the liver of D-Gal/LPS-treated mice express CCR4. Hepatic mononuclear cells were isolated from mice injected intraperitoneally with D-Gal and LPS or with PBS and stained with CCR4, GR1 and CD11b. Dot-plots show the percentage of CCR4-expressing GR1/CD11b-positive cells. One of three representative experiments in which similar results were obtained is shown.
hep26446-sup-0007-suppfig7.tif537KFigure S7. Il-25 reduces the expression of T cell-associated molecules induced by concanavalin (Con) A. A-B. Mice were given i.p. IL-25 (10µg) or vehicle, 1 hour before treatment with ConA. Mice were scarified 6 hours after ConA treatment, livers were explanted and used to measure IFN-γ (Α), IL-17A (B) through Real-Time PCR. Histograms show the mean ± SEM of two experiments in which 6 mice per group were analyzed; *p=0.03; **p=0.01. C-D Mice were treated as described above and sacrificed 24 hours after ConA treatment. Livers were explanted and used to measure IFN-γ (C) and IL-17A (D) by ELISA. Histograms show the mean ± SEM of two experiments in which 6 mice per group were analyzed; *p<0.05.

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