Volume 138, Issue 7, Pages 2499-2508 (June 2010) Signal Transducer and Activator of Transcription 3 Protects From Liver Injury and Fibrosis in a Mouse Model of Sclerosing Cholangitis  Markus Mair, Gernot Zollner, Doris Schneller, Monica Musteanu, Peter Fickert, Judith Gumhold, Christian Schuster, Andrea Fuchsbichler, Martin Bilban, Stefanie Tauber, Harald Esterbauer, Lukas Kenner, Valeria Poli, Leander Blaas, Jan Wilhelm Kornfeld, Emilio Casanova, Wolfgang Mikulits, Michael Trauner, Robert Eferl  Gastroenterology  Volume 138, Issue 7, Pages 2499-2508 (June 2010) DOI: 10.1053/j.gastro.2010.02.049 Copyright © 2010 AGA Institute Terms and Conditions

Figure 1 Severe jaundice and premature lethality in stat3Δhc mdr2−/− mice. (A) Macroscopically, stat3Δhc mdr2−/− livers showed accumulation of bile in the gallbladder (arrow) and bile infarcts (arrowheads). (B) Serum analysis showing high levels of bilirubin in the serum of stat3Δhc mdr2−/− mice indicative for jaundice (n ≥ 4 animals per genotype; age, 7 wk). (C) Kaplan–Meier plot showing premature lethality of stat3Δhc mdr2−/− mice (n = 14 per genotype). Gastroenterology 2010 138, 2499-2508DOI: (10.1053/j.gastro.2010.02.049) Copyright © 2010 AGA Institute Terms and Conditions

Figure 2 Bridging fibrosis in 7-week-old stat3Δhc mdr2−/− mice. (A) H&E-stained sections from stat3flox/flox mdr2−/− and stat3Δhc mdr2−/− mice. Bile infarcts (arrow) and giant hepatocytes (inset in A) were present in stat3Δhc mdr2−/− mice. The fibrotic changes in the periportal areas (arrowheads) strongly were aggravated in stat3Δhc mdr2−/− mice when compared with stat3flox/flox mdr2−/− mice. (B) Chromotrope aniline blue staining showed collagen deposition (blue) in periportal areas (arrowheads) of stat3Δhc mdr2−/− mice. Bridges of collagen connected the periportal areas. Collagen deposition between hepatocytes (chicken-wire fibrosis) was increased in stat3Δhc mdr2−/− mice when compared with stat3flox/flox mdr2−/− mice (insets in B). (C) Periodic acid–Schiff staining for hepatocellular glycogen storage function (glycogen in red) indicated patches of periodic acid–Schiff–negative, nonfunctional hepatocytes in stat3Δhc mdr2−/− mice (arrowheads). (D) Increased fibrosis and liver damage in stat3Δhc mdr2−/− mice was confirmed by quantitative analyses. Fibrosis was quantified by measuring the amount of hydroxyproline (hp) in a liver lobe. Bars represent data from 3 or more animals. alp, alkaline phosphatase; tgl, triglycerides. Gastroenterology 2010 138, 2499-2508DOI: (10.1053/j.gastro.2010.02.049) Copyright © 2010 AGA Institute Terms and Conditions

Figure 3 Stat3 is activated in hepatocytes and cholangiocytes under cholestatic conditions. (A) Immunohistochemical staining for Phospho-Stat3 on sections from stat3flox/flox mdr2−/− mice showed nuclear signals in hepatocytes (arrow) and cholangiocytes (arrowhead) whereas stat3Δhc mdr2−/− mice showed no signals in these cell types. Instead, Kupffer cells (arrowheads) in stat3Δhc mdr2−/− mice displayed strong activation of Stat3, which was confirmed by double-immunohistochemistry for F4/80 (brown) and Phospho-Stat3 (red; upper insets). The lower insets represent images of the fibrotic regions after double-immunohistochemistry for F4/80 (brown) and Phospho-Stat3 (red), indicating that Stat3 also was activated in F4/80-negative nonparenchymal cells (n = 4 animals per genotype; age, 7 wk). *Bile infarct with background staining. (B) Immunohistochemical staining for Phospho-Stat3 on sections from stat3flox/flox and stat3Δhc mice after bile duct ligation. Sham-operated animals displayed no activation of Stat3 (upper images). Bile duct ligation–induced Stat3 activation in hepatocytes (arrow) and cholangiocytes (arrowhead) of stat3flox/flox but not stat3Δhc mice (lower images; n ≥ 6 animals per genotype). (C) Chromotrope aniline blue staining and (D) quantitation of chromotrope aniline blue–stained area (bars, % chromotrope aniline blue–stained area per liver area) showed aggravated collagen deposition (blue) in IL-6−/− mdr2−/− mice when compared with IL-6+/− mdr2−/− littermates (n = 4). (D) Consistently, liver damage parameters were increased. Bars represent data from 4 animals. Gastroenterology 2010 138, 2499-2508DOI: (10.1053/j.gastro.2010.02.049) Copyright © 2010 AGA Institute Terms and Conditions

Figure 4 Stat3 protects from CA-induced hepatic injury. Three-week-old stat3flox/flox and stat3Δhc mice (n ≥ 4 animals per genotype) were fed with 1% CA chow for 28 days and analyzed thereafter. A second experiment yielded similar results. (A) H&E-stained sections revealed severe hepatic injury and formation of bile infarcts (arrowheads) in CA-fed stat3Δhc mice but not in stat3flox/flox mice. (B) Quantitation of bile infarcts in the percentage of liver area (nd, not detectable) on H&E-stained sections. (C and D) Trichrome staining showed deposition of collagen (green; arrowheads) in CA-fed stat3Δhc animals. Bars represent the percentage of trichrome-stained collagen per liver area. (E) Stat3Δhc mice showed increased expression of collagen type I and type III (col I, col III) in response to CA-induced hepatic injury. Bars represent real-time PCR data from 3 or more animals per genotype. (F) Liver damage parameters are increased in CA-fed stat3Δhc mice when compared with stat3flox/flox mice. Bars represent data from 4 or more animals per genotype. alp, alkaline phosphatase. Gastroenterology 2010 138, 2499-2508DOI: (10.1053/j.gastro.2010.02.049) Copyright © 2010 AGA Institute Terms and Conditions

Figure 5 Stat3Δhc mdr2−/− mice show aggravation of cholestasis. (A) Heat map revealing up-regulation of bile acid biosynthesis genes in the absence of Stat3. (B) Bile acid (ba) concentrations in the serum are increased in the absence of Stat3 (n ≥ 3 animals per genotype; age, 7 wk). (C) Real-time PCR analysis showing up-regulation of IL-6 and TNFα expression in stat3Δhc mdr2−/− mice (n ≥ 3 animals per genotype; age, 7 wk). (D) Enzyme-linked immunosorbent analysis for TNFα protein in liver lysates (n = 4; age, 7 wk). (E) Electrophoretic mobility shift assay for NF-κB activity. Whole-liver lysates from stat3Δhc mdr2−/− and stat3flox/flox mdr2−/− mice were used for electrophoretic mobility shift assay (lanes 1–6). Competition assays were performed by adding unlabeled oligonucleotides in 25-fold molar excess (lanes 7 and 8). As control, HEK 293 cells were transfected with plasmids expressing p65 (lane 9). Supershift assays were performed by preincubation with 1 μg (lane 10) and 4 μg (lane 11) p65 antibody to the reaction mixture. (F) Double immunofluorescence of liver sections showed localization of activated Phospho-Ser-536-p65 (green) in F4/80+ Kupffer cells (red; arrowheads in the merged column) of stat3Δhc mdr2−/− mice (n = 4; age, 7 wk). Gastroenterology 2010 138, 2499-2508DOI: (10.1053/j.gastro.2010.02.049) Copyright © 2010 AGA Institute Terms and Conditions

Figure 6 Down-regulation of signaling pathways that protect from bile acid–induced hepatic injury in stat3Δhc mice. (A) Heat map revealing down-regulation of IGF-1 and EGFR in liver samples lacking Stat3. (B) Confirmation of reduced mRNA expression for IGF-1 and EGFR in liver samples of stat3Δhc mice, stat3Δhc mdr2−/− mice, and stat3Δhc mice after CA feeding by real-time PCR (n ≥ 3 animals per genotype; age, 7 wk). (C) Western blot data showing reduced protein expression for EGFR and IGF-1 in stat3Δhc mice and stat3Δhc mdr2−/− mice when compared with controls. The substantial amount of P-Stat3 in stat3Δhc mdr2−/− mice is most likely owing to activation of Stat3 in Kupffer cells and other nonparenchymal cells. Expression of Hsc-70 was used as loading control. (D) Model of how Stat3 may protect from bile acid–induced hepatic injury. For details see the Discussion section. bd, bile duct; ba, bile acids. Gastroenterology 2010 138, 2499-2508DOI: (10.1053/j.gastro.2010.02.049) Copyright © 2010 AGA Institute Terms and Conditions