Caspase-8 is a gender-specific modulator of chronic cholestatic liver disease in female Mdr2 knockout mice

Background and Aims:

Females are more susceptible to hepatic cholestasis but the underlying mechanisms are incompletely understood. Mdr2 knockout (Mdr2^-/-) mice are a well-established animal model of human primary sclerosing cholangitis (PSC) also comprising enhanced fibrosis susceptibility in female mice. We aimed to investigate the impact of extrinsic apoptosis in PSC by deleting Caspase-8 in hepatocytes of Mdr2^-/- mice.

Methods:

Mdr2^-/-Casp8^Δhepa double knockout mice were generated in a C57Bl/6 background by crossing Mdr2^-/- mice with animals lacking Caspase-8 in hepatocytes (Casp8^Δhepa). Cre-negative littermates (Mdr2^-/-Casp8^f/f) and C57Bl/6 WT mice were used as reference. Male and female animals were analysed for liver fibrosis by quantification of hepatic collagen deposition at the age of 12, 26 and 52 weeks. Expression of genes related to inflammation, cell death, proliferation and cancer was determined by qPCR.

Results:

In good agreement with earlier studies we detected increased liver fibrosis in female compared to male Mdr2^-/- mice at the age of 26 – 52 weeks. Unexpectedly, deletion of Caspase-8 in male Mdr2^-/- mice did not affect the degree of liver fibrosis at any age investigated. However, in female Mdr2^-/- mice at the age of 26 weeks, concomitant inactivation of Caspase-8 triggered approximately 2fold increased gene expression of Tumor Necrosis Factor alpha (TNF) and Receptor-interacting serine/threonine-protein kinase 3 (RIPK3). Of note, both genes are involved in regulating inflammation and programmed cell death including apoptosis and necroptosis. In contrast, basal TNF and RIPK3 gene expression was not different between male and female WT or Mdr2^-/- mice suggesting that the observed gender disparity observed in Mdr2^-/-Casp8^Δhepa mice was provoked specifically by Caspase-8 deletion. In addition, we found a significant correlation between the fibrosis level in 26 week old Mdr2^-/-Casp8^Δhepa mice and gene expression levels of RIPK3, Cyclin E2 (involved in cell cycle regulation) and c-myc (related to fibrosis and cancer). In agreement with these findings, loss of Caspase-8 significantly enhanced liver fibrosis exclusively in female Mdr2^-/- mice at the age of 52 weeks reflecting a stage of advanced disease progression.

Conclusions:

Caspase-8 is dispensable for initiation and progression of liver injury in male Mdr2^-/- mice. However, ablation of Caspase-8 triggers significantly elevated liver fibrosis in female Mdr2^-/- mice which precedes enhanced gene expression of TNF and RIPK3. Thus Caspase-8 can be considered as a gender-specific modulator of death pathways in the cholestatic liver.