title: Role of interleukin-1 and its antagonism of hepatic stellate cell proliferation and liver fibrosis in the Abcb4(-/-) mouse model.
abstract: OBJECTIVE
  To study the interleukin-1 (IL-1) pathway as a therapeutic target for liver fibrosis in vitro and in vivo using the ATP-binding cassette transporter b4(-/-) (Abcb4(-/-)) mouse model.
  
  
  METHODS
  Female and male Abcb4(-/-) mice from 6 to 13 mo of age were analysed for the degree of cholestasis (liver serum tests), extent of liver fibrosis (hydroxyproline content and Sirius red staining) and tissue-specific activation of signalling pathways such as the IL-1 pathway [quantitative polymerase chain reaction (qPCR)]. For in vivo experiments, murine hepatic stellate cells (HSCs) were isolated via pronase-collagenase perfusion followed by density gradient centrifugation using female mice. Murine HSCs were stimulated with up to 1 ng/mL IL-1β with or without 2.5 μg/mL Anakinra, an IL-1 receptor antagonist, respectively. The proliferation of murine HSCs was assessed via the BrdU assay. The toxicity of Anakinra was evaluated via the fluorescein diacetate hydrolysis (FDH) assay. In vivo 8-wk-old Abcb4(-/-) mice with an already fully established hepatic phenotype were treated with Anakinra (1 mg/kg body-weight daily intraperitoneally) or vehicle and liver injury and liver fibrosis were evaluated via serum tests, qPCR, hydroxyproline content and Sirius red staining.
  
  
  RESULTS
  Liver fibrosis was less pronounced in males than in female Abcb4(-/-) animals as defined by a lower hydroxyproline content (274 ± 64 μg/g vs 436 ± 80 μg/g liver, respectively; n = 13-15; P < 0.001; Mann-Whitney U-test) and lower mRNA expression of the profibrogenic tissue inhibitor of metalloproteinase-1 (TIMP) (1 ± 0.41 vs 0.66 ± 0.33 fold, respectively; n = 13-15; P < 0.05; Mann-Whitney U-test). Reduced liver fibrosis was associated with significantly lower levels of F4/80 mRNA expression (1 ± 0.28 vs 0.71 ± 0.41 fold, respectively; n = 12-15; P < 0.05; Mann-Whitney U-test) and significantly lower IL-1β mRNA expression levels (1 ± 0.38 vs 0.44 ± 0.26 fold, respectively; n = 13-15; P < 0.001; Mann-Whitney U-test). No gender differences in the serum liver parameters [bilirubin; alanine aminotransferase (ALT); aspartate aminotransferase and alkaline phosphatase (AP)] were found. In vitro, the administration of IL-1β resulted in a significant increase in HSC proliferation [0.94 ± 0.72 arbitrary units (A.U.) in untreated controls, 1.12 ± 0.80 A.U. at an IL-1β concentration of 0.1 ng/mL and 1.18 ± 0.73 A.U. at an IL-1β concentration of 1 ng/mL in samples from n = 6 donor animals; P < 0.001; analyses of variance (ANOVA)]. Proliferation was reduced significantly by the addition of 2.5 μg/mL Anakinra (0.81 ± 0.60 A.U. in untreated controls, 0.92 ± 0.68 A.U. at an IL-1β concentration of 0.1 ng/mL, and 0.91 ± 0.69 A.U. at an IL-1β concentration of 1 ng/mL; in samples from n = 6 donor animals; P < 0.001; ANOVA) suggesting an anti-proliferative effect of this clinically approved IL-1 receptor antagonist. The FDH assay showed this dose to be non-toxic in HSCs. In vivo, Anakinra had no effect on the hepatic hydroxyproline content, liver serum tests (ALT and AP) and pro-fibrotic (collagen 1α1, collagen 1α2, transforming growth factor-β, and TIMP-1) and anti-fibrotic [matrix metalloproteinase 2 (MMP2), MMP9 and MMP13] gene expression after 4 wk of treatment. Furthermore, the hepatic IL-1β and F4/80 mRNA expression levels were unaffected by Anakinra treatment.
  
  
  CONCLUSIONS
  IL-1β expression is associated with the degree of liver fibrosis in Abcb4(-/-) mice and promotes HSC proliferation. IL-1 antagonism shows antifibrotic effects in vitro but not in Abcb4(-/-) mice.
authors.affiliation: Florian P Reiter, Ralf Wimmer, Lena Wottke, Renate Artmann, Jutta M Nagel, Alexander L Gerbes, Simon Hohenester, Gerald U Denk, Department of Medicine II, Liver Center Munich, University of Munich, D-81377 Munich, Germany.
authors.forename: Florian P
authors.initials: FP
authors.lastname: Reiter
authors.affiliation: Florian P Reiter, Ralf Wimmer, Lena Wottke, Renate Artmann, Jutta M Nagel, Alexander L Gerbes, Simon Hohenester, Gerald U Denk, Department of Medicine II, Liver Center Munich, University of Munich, D-81377 Munich, Germany.
authors.forename: Ralf
authors.initials: R
authors.lastname: Wimmer
authors.affiliation: Florian P Reiter, Ralf Wimmer, Lena Wottke, Renate Artmann, Jutta M Nagel, Alexander L Gerbes, Simon Hohenester, Gerald U Denk, Department of Medicine II, Liver Center Munich, University of Munich, D-81377 Munich, Germany.
authors.forename: Lena
authors.initials: L
authors.lastname: Wottke
authors.affiliation: Florian P Reiter, Ralf Wimmer, Lena Wottke, Renate Artmann, Jutta M Nagel, Alexander L Gerbes, Simon Hohenester, Gerald U Denk, Department of Medicine II, Liver Center Munich, University of Munich, D-81377 Munich, Germany.
authors.forename: Renate
authors.initials: R
authors.lastname: Artmann
authors.affiliation: Florian P Reiter, Ralf Wimmer, Lena Wottke, Renate Artmann, Jutta M Nagel, Alexander L Gerbes, Simon Hohenester, Gerald U Denk, Department of Medicine II, Liver Center Munich, University of Munich, D-81377 Munich, Germany.
authors.forename: Jutta M
authors.initials: JM
authors.lastname: Nagel
authors.affiliation: Florian P Reiter, Ralf Wimmer, Lena Wottke, Renate Artmann, Jutta M Nagel, Alexander L Gerbes, Simon Hohenester, Gerald U Denk, Department of Medicine II, Liver Center Munich, University of Munich, D-81377 Munich, Germany.
authors.forename: Manuel O
authors.initials: MO
authors.lastname: Carranza
authors.affiliation: Florian P Reiter, Ralf Wimmer, Lena Wottke, Renate Artmann, Jutta M Nagel, Alexander L Gerbes, Simon Hohenester, Gerald U Denk, Department of Medicine II, Liver Center Munich, University of Munich, D-81377 Munich, Germany.
authors.forename: Doris
authors.initials: D
authors.lastname: Mayr
authors.affiliation: Florian P Reiter, Ralf Wimmer, Lena Wottke, Renate Artmann, Jutta M Nagel, Alexander L Gerbes, Simon Hohenester, Gerald U Denk, Department of Medicine II, Liver Center Munich, University of Munich, D-81377 Munich, Germany.
authors.forename: Christian
authors.initials: C
authors.lastname: Rust
authors.affiliation: Florian P Reiter, Ralf Wimmer, Lena Wottke, Renate Artmann, Jutta M Nagel, Alexander L Gerbes, Simon Hohenester, Gerald U Denk, Department of Medicine II, Liver Center Munich, University of Munich, D-81377 Munich, Germany.
authors.forename: Peter
authors.initials: P
authors.lastname: Fickert
authors.affiliation: Florian P Reiter, Ralf Wimmer, Lena Wottke, Renate Artmann, Jutta M Nagel, Alexander L Gerbes, Simon Hohenester, Gerald U Denk, Department of Medicine II, Liver Center Munich, University of Munich, D-81377 Munich, Germany.
authors.forename: Michael
authors.initials: M
authors.lastname: Trauner
authors.affiliation: Florian P Reiter, Ralf Wimmer, Lena Wottke, Renate Artmann, Jutta M Nagel, Alexander L Gerbes, Simon Hohenester, Gerald U Denk, Department of Medicine II, Liver Center Munich, University of Munich, D-81377 Munich, Germany.
authors.forename: Alexander L
authors.initials: AL
authors.lastname: Gerbes
authors.affiliation: Florian P Reiter, Ralf Wimmer, Lena Wottke, Renate Artmann, Jutta M Nagel, Alexander L Gerbes, Simon Hohenester, Gerald U Denk, Department of Medicine II, Liver Center Munich, University of Munich, D-81377 Munich, Germany.
authors.forename: Simon
authors.initials: S
authors.lastname: Hohenester
authors.affiliation: Florian P Reiter, Ralf Wimmer, Lena Wottke, Renate Artmann, Jutta M Nagel, Alexander L Gerbes, Simon Hohenester, Gerald U Denk, Department of Medicine II, Liver Center Munich, University of Munich, D-81377 Munich, Germany.
authors.forename: Gerald U
authors.initials: GU
authors.lastname: Denk
keywords: Cholestasis; Interleukin-1; Liver fibrosis; Primary sclerosing cholangitis; The ATP-binding cassette transporter b4