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hep26429-sup-0001-suppfig1.tif1561KSupplementary Figure 1: Purity of F4/80-positive hepatic macrophages. A-B. Purity of MACS-sorted F4/80-positive hepatic macrophages was determined by FACS analysis for F4/80 (A) and by phagocytosis of fluorogenic pHrodo Bioparticles that selectively emit fluorescence after phagocytosis (B). C. M1 and M2 profiles in HM from sham and BDL livers were assessed by qPCR for M1 markers iNos and Cox2, and M2 markers Fizz-1 and Ym1 (left panel). Thioglycollate-elicited peritoneal macrophages treated 16 hours with LPS (10 ng/mL) and interferon gamma (10 ng/mL) were used as positive control for the M1 profile (right panel). Thioglycollate-elicited peritoneal macrophages treated 16 hours with IL-4 (10 ng/mL) and IL-10 (10 ng/mL) were used as positive control for M2 profile (right panel).
hep26429-sup-0002-suppfig2.tif2700KSupplementary Figure 2. IPA pathway analysis from microarrays HSCs co-cultured with hepatic macrophages. A. Displayed are the 20 most significant categories of IPA tox functions. B. Displayed are the 20 most significant categories of IPA biological functions.
hep26429-sup-0003-suppfig3.tif2718KSupplementary Figure 3. Inhibition of NF-κB-dependent gene expression by IKK inhibitor BAY 11-7082. A-B. mRNA expression of HSC activation markers or NF-κB responsive genes was determined after treatment with rmIL-1β (5 ng/ml) for 3h (A) and 24 hours (B) in the presence or absence of BAY 11-70-82 (1 µM). C-D. mRNA expression of HSC activation markers or NF-κB responsive genes was determined by qPCR in HSC cultured with HM supernatant in the presence or absence of BAY 11-7082 (1µM) for 4h (C) and 24h (D).
hep26429-sup-0004-suppfig4.tif3252KSupplementary Figure 4. Macrophage depletion reduces CCl4- and BDL-induced liver fibrosis. A-D. Mice underwent BDL for 15 days and were injected with vehicle or clodronate as depicted (A). Hepatic fibrosis was determined by Picrosirius red staining and morphometric analysis using polarized light for vehicle-treated (n=14) and clodronate- treated (n=10) mice (B), and by hepatic expression of fibrogenic genes using qPCR (C). Depletion of F4/80-positive liver macrophages was confirmed by F4/80 immunohistochemistry (D). E-H. Mice received clodronate or vehicle injections during CCl4-induced hepatic fibrosis as depicted (E) with the CCL4 dose for day 1 and 5 being 0.125μl/g and 0.25μl/g for the remaining injections. Hepatic fibrosis in vehicle-treated (n=10) and clodronate-treated (n=8) mice was determined by Picrosirius red staining and morphometric analysis using  polarized light (F) and by hepatic expression of fibrogenic genes using qPCR (G). Depletion of F4/80-positive liver macrophages was confirmed by F4/80 immunohistochemistry (H). I. Bile duct ligated mice were injected every 5 days for a total of 3 injections with PBS (n=4) or clodronate (n=3) followed by quantification of F4/80- CD11b- double positive macrophages using flow cytometry FACS plots show percentage of double positive cells, the bar graph shows cell numbers per gram liver. *p<0.05 ** p<0.01
hep26429-sup-0005-suppfig5.tif750KSupplementary Figure 5. IL-1β and TNFα do not contribute to HSC activation HSCs were serum starved in 0.1% FBS in the presence or absence of hepatic macrophages, and the absence or presence of rmIL-1β (5 ng/ml). Cell death was determined by propidium iodide staining (red). Nuclei were visualized by Hoechst staining (blue). *p<0.05; **p<0.01.
hep26429-sup-0006-suppfig6.tif2340KSupplementary Figure 6. IL-1 receptor deficient mice do not display reduced liver fibrosis in multiple fibrosis models. A-D. Wild-type and IL-1R-deficient mice were subjected to fibrosis induction by bile duct ligation for 3 weeks (A, n=11 wild-type mice and n=7 IL-1R knockout mice), treatment with 8 injections of CCl4 (B, n=10 mice/group), treatment with 20 injections of CCl4 (C, n=8 mice/group), or 18 weeks of thioacetamide in drinking water (D, 15 wild-type mice and 12 IL-1Rko mice). Hepatic fibrosis was quantified by Sirius Red staining and morphometry.
hep26429-sup-0007-suppfig7.tif1251KSupplementary Figure 7. Hepatic macrophages upregulate Trail decoy receptor expression in hepatic stellate cells. A. mRNA expression of Trail decoy receptors Tnfrsf22 and Tnfrsf23 was determined qPCR in HSC co-cultured with HM in the presence or absence of an antagonistic TNFRI-Fc chimera, an antagonistic IL-1RI-Fc chimera or both. B. Expression of Trail decoy receptors Tnfrsf22 and Tnfrsf23 was determined by qPCR in ultrapure unplated HSCs isolated from 15 day bile duct-ligated vehicle-treated mice (n=6 isolations) and clodronate-treated mice (n=4 isolations) by a combination of gradient centrifugation and FACS analysis without additional plating. Data is expressed as fold induction in comparison to quiescent HSCs isolated from control wt mice (n=3 isolations), or as ratio between the investigated mRNA and 18s mRNA if mRNA expression was not detectable in qHSC. *p<0.05; **p<0.01.
hep26429-sup-0008-suppfig8.tif3206KSupplementary Figure 8. Depletion of dendritic cells by CD11c-DTR and 120-G8 antibody-mediated ablation. A. Livers of PBS- and DT-treated chimeric CD11c-DTR-eGFP mice after 4 weeks of fibrosis induction was harvested, and freshly isolated intrahepatic leukocytes where flox-cytometerically analyzed for frequency of cDC. B. Liver of C57Bl/6 mice after 4 weeks of CCL4 fibrogenesis, with or without 120G8 antibody treatment, were analyzed flow-cytometrically for the frequency pDC. C. Spleens from PBS- and DT-treated CD11c-DTR-eGFP chimeric mice were harvested 24h after the last injection. Splenocytes were isolated and CD11c-driven eGFP expression was determined by flow cytometry.
hep26429-sup-0009-suppfig9.tif653KSupplementary Figure 9. Neutrophilia in mice with dendritic cell ablation by CD11c-DTR. CD11c-DTR chimeric mice received DT treatment as described in the Methods. 24 hours after the last DT injection, livers were collected and hepatic neutrophil numbers was determined by flow cytometry using Ly6G and CD11b. FACS plots show percentage of double positive cells, the bar graph shows cell numbers per gram liver. *p<0.05, * p<0.01
hep26429-sup-0010-supptable1.xls747KSupplementary Table 1. Genes with more than 2-fold and significant up- or downregulation induced by co-culture of HSCs with hepatic macrophages. Shown are all probe sets with more than 2-fold up- or downregulation after co-culture with hepatic macrophages for 5 days. Displayed are the fold induction, p-value and adjusted p-value. In addition, comparison of activated HSCs, activated HSCs co-cultured with hepatic macrophages, bile duct ligation activated HSCs and CCl4-activated HSCs with quiescent HSCs are included for each probe set.
hep26429-sup-0011-supptable2.xls59KSupplementary Table 2. List of NF-κB genes with more than 10-fold upregulation in HSCs after co-culture with hepatic macrophages. Shown is a list of genes with more than 10-fold induction in HSCs after co-culture with hepatic macrophages for 5 days. Genes that are NF-κB dependent are annotated with the relevant data source. Shown is the fold induction.
hep26429-sup-0012-suppInfo.doc105KSupplementary Information.

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