An improved droplet digital PCR assay reveals the stability of intrahepatic hepatitis B virus cccDNA.

The persistence of covalently closed circular DNA (cccDNA) poses a major obstacle to curing chronic hepatitis B (CHB). Here, we used droplet digital PCR (ddPCR) for cccDNA quantitation. ddPCR measured a less than two-fold difference in the intrahepatic cccDNA content more accurately than conventional real-time PCR (qPCR), (R2=0.9416 and R2=0.8963, respectively) and had also higher sensitivity and specificity than qPCR. The results of ddPCR correlated closely with serum HB core-related antigen and HB surface antigen (HBsAg) in 24 HBV-infected human-liver-chimeric mice (PXB-mice). We demonstrated that the total cccDNA content did not change during liver repopulation, although the cccDNA content per hepatocyte was reduced in PXB-mice after entecavir treatment. In the 6 patients with HBV-related hepatocellular carcinoma, ddPCR detected cccDNA in both tumor and non-tumor tissues. In 13 HBeAg-negative CHB patients with pegylated interferon alpha-2a, cccDNA contents from paired biopsies were more significantly reduced in virological response (VR) than in non-VR at week 48 (p=0.0051). Interestingly, cccDNA levels were the lowest in VR with HBsAg clearance but remained detectable after the treatment. Collectively, ddPCR revealed that cccDNA content is stable during hepatocyte proliferation and persists at quantifiable levels, even after serum HBsAg clearance.

β-Catenin-NFkB-CFTR interactions in cholangiocytes regulate inflammation and fibrosis during ductular reaction

Expansion of biliary epithelial cells (BECs) during ductular reaction (DR) is observed in liver diseases including cystic fibrosis (CF), and associated with inflammation and fibrosis, albeit without complete understanding of underlying mechanism. Using two different genetic mouse knockouts of b-catenin, one with b-catenin loss is hepatocytes and BECs (KO1), and another with loss in only hepatocytes (KO2), we demonstrate disparate long-term repair after an initial injury by 2-week choline-deficient ethionine-supplemented diet. KO2 show gradual liver repopulation with BEC-derived b-catenin-positive hepatocytes, and resolution of injury. KO1 showed persistent loss of b-catenin, NF-kB activation in BECs, progressive DR and fibrosis, reminiscent of CF histology. We identify interactions of b-catenin, NFkB and CF transmembranous conductance regulator (CFTR) in BECs. Loss of CFTR or b-catenin led to NF-kB activation, DR and inflammation. Thus, we report a novel b-catenin-NFkB-CFTR interactome in BECs, and its disruption may contribute to hepatic pathology of CF.

β-Catenin-NFκB-CFTR interactions in cholangiocytes regulate inflammation and fibrosis during ductular reaction

Expansion of biliary epithelial cells (BECs) during ductular reaction (DR) is observed in liver diseases including cystic fibrosis (CF), and associated with inflammation and fibrosis, albeit without complete understanding of underlying mechanism. Using two different genetic knockouts of β-catenin, one with ß-catenin loss is hepatocytes and BECs (KO1), and another with loss in only hepatocytes (KO2), we demonstrate disparate long-term repair after an initial injury by 2-week choline-deficient ethionine supplemented diet. KO2 show gradual liver repopulation with BEC-derived β-cateninpositive hepatocytes, and resolution of injury. KO1 showed persistent loss of β-catenin, NF-κB activation in BECs, progressive DR and fibrosis, reminiscent of Cystic fibrosis (CF) histology. We identify interactions of β-catenin, NFκB and Cystic fibrosis transmembranous conductance regulator (CFTR) in BECs. Loss of CFTR or β-catenin led to NF-κB activation, DR and inflammation. Thus, we report novel β-catenin-NFκBCFTR interactome in BECs, and its disruption may contribute to hepatic pathology of CF.

Highly Accurate, Specific And Sensitive Quantitation By Droplet Digital PCR Reveals The Stability of Intrahepatic Hepatitis B Virus cccDNA

The persistence of covalently closed circular DNA (cccDNA) poses a major obstacle to curing chronic hepatitis B (CHB). Here, we used droplet digital PCR (ddPCR) for cccDNA quantitation. ddPCR measured a less than two-fold difference in the intrahepatic cccDNA content more accurately than conventional real-time PCR (qPCR), (R2=0.9416 and R2=0.8963, respectively) and had also higher sensitivity and specificity than qPCR. The results of ddPCR correlated more closely with serum HB core-related antigen (R2=0.9843) than HB surface antigen (HBsAg) (R2=0.9742) in 24 HBV-infected human-liver-chimeric mice (PXB-mice). We demonstrated that the total cccDNA content did not change during liver repopulation, although the cccDNA content per hepatocyte was reduced in PXB-mice after entecavir treatment. In the 6 patients with HBV-related hepatocellular carcinoma, ddPCR detected cccDNA in both tumor and non-tumor tissues. In 13 HBeAg-negative CHB patients with pegylated interferon alpha-2a, cccDNA contents from paired biopsies were more significantly reduced in virological response (VR) than in non-VR at week 48 (p=0.0051). Interestingly, cccDNA levels were the lowest in VR with HBsAg clearance but remained detectable after the treatment. Collectively, ddPCR revealed that cccDNA content is stable during hepatocyte proliferation and persists at quantifiable levels, even after serum HBsAg clearance.

Therapeutic liver repopulation by transient acetaminophen selection of gene-modified hepatocytes

Gene therapy by integrating vectors is promising for monogenic liver diseases, especially in children where episomal vectors remain transient. However, reaching the therapeutic threshold with genome-integrating vectors is challenging. Therefore, we developed a method to expand hepatocytes bearing therapeutic transgenes. The common fever medicine acetaminophen becomes hepatotoxic via cytochrome p450 metabolism. Lentiviral vectors with transgenes linked in cis to a Cypor shRNA were administered to neonatal mice. Hepatocytes lacking the essential cofactor of Cyp enzymes, NADPH-cytochrome p450 reductase (Cypor), were selected in vivo by acetaminophen administration, replacing up to 50% of the hepatic mass. Acetaminophen treatment of the mice resulted in over 30-fold expansion of transgene-bearing hepatocytes and achieved therapeutic thresholds in hemophilia B and phenylketonuria. We conclude that therapeutically modified hepatocytes can be selected safely and efficiently in preclinical models with a transient regimen of moderately hepatotoxic acetaminophen.

Liver homeostasis is maintained by midlobular zone 2 hepatocytes

The liver is organized into zones in which hepatocytes express different metabolic enzymes. The cells most responsible for liver repopulation and regeneration remain undefined, because fate mapping has only been performed on a few hepatocyte subsets. Here, 14 murine fate-mapping strains were used to systematically compare distinct subsets of hepatocytes. During homeostasis, cells from both periportal zone 1 and pericentral zone 3 contracted in number, whereas cells from midlobular zone 2 expanded in number. Cells within zone 2, which are sheltered from common injuries, also contributed to regeneration after pericentral and periportal injuries. Repopulation from zone 2 was driven by the insulin-like growth factor binding protein 2–mechanistic target of rapamycin–cyclin D1 (IGFBP2-mTOR-CCND1) axis. Therefore, different regions of the lobule exhibit differences in their contribution to hepatocyte turnover, and zone 2 is an important source of new hepatocytes during homeostasis and regeneration.