Combined accumulation of hepatitis B surface antigen and mutated alpha-1 antitrypsin promotes the development of liver disease via autophagy overload and activation of p62-mTOR-Nrf2 axis

Background & aim: Liver constitutes a major protein secreting organ and because of that, accumulation of secretory proteins in the endoplasmatic reticulum (ER) constitutes a hallmark of multiple liver disorders. Among them, aggregation of mutated α1-antitrypsin termed as PiZ is a characteristic feature of AAT deficiency, whereas retention of hepatitis B surface protein (HBs) is commonly found in chronic hepatitis B infection and gives rise to ground glass hepatocytes. To study the effect of these proteotoxic situations, we crossbred PiZ and HBs overexpressing animals.

Aims & methodology: Mouse and human livers with combined PiZ/HBs retention were evaluated by quantitative RT-PCR, immunoblotting, histological/immunological staining and biochemical assays. DHE staining was employed to visualize oxidative stress and p62-containing aggregates were isolated via MACS. Crossbreeding of PiZ mice with CHOP knockouts was performed to address the role of CHOP in proteotoxic injury.

Results: HBs-PiZ mice were viable and developed normally. At 2 months of age, they displayed a significantly stronger liver injury (ALT: PiZ-HBs 88, HBs 53, PiZ 32, p < 0.05 for both). While both AAT and HBs were retained in the ER, both in human and the mice, the inclusions displayed a distinct, non-overlapping pattern. Among 10 months old animals, PiZ-HBs mice displayed a more pronounced dysplastic changes, liver fibrosis and higher hepatocyte proliferation. At 14 months of age, double transgenic animals developed larger tumor nodules (Double 141, HBs 36, PiZ 3.7 mm2, p < 0.05 for both) and a higher tumor load. Analysis of 16 gene expression signature revealed a more aggressive tumor subtype in HBs-PiZ mice as evidenced by down-regulation of Cyp2e1, Aqp9, Apoc4, (p < 0.001 for all) and C1 s (p < 0.05). As the presumable drivers of liver phenotype, HBs-PiZ animals exhibited a marked accumulation of the autophagy adaptor protein p62 that was observed in the protein overloaded hepatocytes. Moreover, an activation of p62-mTOR axis with increased pmTORS2448 and p4EBP1 levels was detected. p62 MACS uncovered a precipitation of otherwise soluble HBs and accumulation of ubiquitinated proteins in the double-transgenic animals, but not in HBs mice. As a result of increased proteotoxic stress, an accumulation of reactive oxygen species and an activation of Nrf2 signalling was detected in HBs-PiZ animals. NF-kB activation along with strong CHOP overexpression evidenced an activation of ER overload response. However, this response does not seem to contribute to the observed phenotype, since the ablation of CHOP did not alter the extent of liver injury and fibrosis in 19 months old PiZ/PiZ-CHOP-KO mice.

Conclusions: Our results suggest that a combined accumulation of PiZ and HBs accelerates the development of liver injury due to autophagy overload with subsequent activation of p62-mTOR-Nrf2 axis.