Role of interleukin-1 and its antagonism of hepatic stellate cell proliferation and liver fibrosis in the Abcb4-/- mouse model

AIM: 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-1 beta with or without 2.5 mu g/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 mu g/g vs 436 +/- 80 mu g/g liver, respectively; n = 13-15; P < 0.001; MannWhitney 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-1 beta 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-1 beta 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-1 beta concentration of 0.1 ng/mL and 1.18 +/- 0.73 A.U. at an IL-1 beta 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 mu g/mL Anakinra (0.81 +/- 0.60 A.U. in untreated controls, 0.92 +/- 0.68 A.U. at an IL-1 beta concentration of 0.1 ng/mL, and 0.91 +/- 0.69 A.U. at an IL-1 beta 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 profibrotic (collagen 1 alpha 1, collagen 1 alpha 2, transforming growth factor-beta, and TIMP-1) and anti-fibrotic [matrix metalloproteinase 2 (MMP2), MMP9 and MMP13] gene expression after 4 wk of treatment. Furthermore, the hepatic IL-1 beta and F4/80 mRNA expression levels were unaffected by Anakinra treatment. CONCLUSION: IL-1 beta 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.