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hep26697-sup-0001-suppfig1.tif3816KSupplementary Figure 1. Expression of CCR6 and CCL20 in different disease etiologies. A+B RNA was extracted from liver samples of patients with chronic liver disease and control tissue. Only patients with cirrhosis were included in the analysis. CCR6 (A) and CCL20 (B) expression levels were measured by quantitative real-time PCR (qPCR), normalized to the housekeeping gene β-actin. C Liver sections of patients with biliary diseases were stained for CCL20; representative pictures from n=3 individual patients per group are shown. Scale bar: 100μm. Viral: viral hepatitis, PBC: primary biliary cirrhosis, PSC: primary sclerosing cholangitis, alcohol: alcoholic liver disease. */# P<0.05 **/## P<0.01 ***/### P<0.001. #=compared to healthy controls.
hep26697-sup-0002-suppfig2.tif2859KSupplementary Figure 2. Ccr6 and Ccl20 are up-regulated in murine chronic liver disease. A-B Wt mice were treated with CCl4 thrice weekly over 4 weeks, and RNA was extracted from liver. Ccr6 and Ccl20 expression were measured by qPCR. C Primary hepatocytes were isolated from control or CCl4-treated wt mice and plated on cell culture dishes. Representative pictures, taken immediately after isolation. D Hepatocytes (Hepa), hepatic stellate cells (HSC) and macrophages (MΦ) were isolated from livers of control or CCl4-treated wt mice, and Ccl20 expression was determined by qPCR. nd= not detectable, *P<0.05 **P<0.01 ***P<0.001 (U-test for human data, t-test for murine data).
hep26697-sup-0003-suppfig3.tif1011KSupplementary Figure 3. No difference in relative composition of hepatic immune cells between wt and Ccr6-/- mice. A Wt and Ccr6-/- mice were treated thrice weekly with CCl4 for 4 weeks. Cells were isolated from liver and stained for CD45 to identify leukocytes, dead cells were excluded by Hoechst 33258. Leukocytes were stained with Ly6G for neutrophils, CD11b and F4/80 for macrophages, CD4 and CD8 for T-cells, NK1.1 to identify NK-cells, and B220 for B-cells. Relative amounts of the individual immune cell subpopulations as fractions of living CD45+ cells are shown.
hep26697-sup-0004-suppfig4.tif833KSupplementary Figure 4. Flow cytometric analysis of hepatic T-helper cells. A Wt and Ccr6-/- mice were treated thrice weekly with CCl4 for 4 weeks, and total RNA was isolated from liver tissue. Expression of transcription factors for CD4 T-helper cell subtypes was measured by qPCR, namely T-bet for Th1 cells, Gata3 for Th2 cells, Foxp3 for Treg, and Rorγt for Th17 cells. B Leukocytes were isolated from livers of wt and Ccr6-/- mice after CCl4 treatment or control mice and stained for CD3, CD4 and subsequently T-bet, FoxP3 and RORγt to identify T-helper cell subsets. Relative amounts of CD4+ T-cells are shown. C Representative FACS plots of same CD4+ T-cell subsets as in B. Dashed histogram: isotype control, grey histogram: Ccr6-/-, black histogram: wt. ns: not significant, *P<0.05 **P<0.01 ***P<0.001.
hep26697-sup-0005-suppfig5.tif4505KSupplementary Figure 5. γδ T-cells inhibit myofibroblast activation and induce apoptosis in GRX cells. A-C Hepatic CD4+ T-cells, γδ T-cells and NK-cells were isolated by FACS sorting from wt mice 48h after single CCl4 injection. Either isolated cells or supernatant from overnight cultures were incubated with the hepatic stellate cell (HSC)-line GRX for 48h (positive control: GRX-cells cultured with 5ng/ml TGF-β1, negative control: untreated cells). A Representative pictures of co-cultures and quantification of cell density in cultures. Scale bar: 200μm. B Gene expression of Collagen1 and Pdgfrβ. C GRX cells were fixed, permeabilized and stained with propidium iodide (PI) for FACS analysis. Amounts of apoptotic cells are shown as percent of control. D Hepatic CD4+ T-cells, γδ T-cells and NK-cells were isolated from wt mice and analysed for expression of Fas ligand (FasL). Representative FACS plots of γδ T-cells are shown. E Hepatic γδ T-cells were isolated by FACS sorting from wt mice 48h after a single CCl4 injection and incubated with primary HSC, isolated from wt mice, for 48h, with or without anti-IL-22 antibody or control IgG. HSC were fixed, permeabilized and stained with propidium iodide (PI) for FACS analysis. Representative histograms with sub-G1 cells are shown. Amounts of apoptotic cells are shown as percent of control. *P<0.05 **P<0.01 ***P<0.001. Data are expressed as mean±SEM from two independent experiments.
hep26697-sup-0006-suppinfo.doc72KSupporting Information

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