Liver repopulation with xenogenic hepatocytes in B and T cell-deficient mice leads to chronic hepadnavirus infection and clonal growth of hepatocellular carcinoma

Abstract

To investigate host and viral mechanisms determining hepadnaviral persistence and hepatocarcinogenesis, we developed a mouse model by transplanting woodchuck hepatocytes into the liver of mice that contain the urokinase-type plasminogen activator transgene (uPA) and lack mature B and T lymphocytes due to a recombination activation gene 2 (RAG-2) gene knockout. The woodchuck hepatocytes were transplanted via intrasplenic injection and were found to integrate into the recipient mouse liver cord structure. Normal adult woodchuck hepatocytes proliferated and reconstituted up to 90% of the uPA͞RAG-2 mouse liver. uPA͞RAG-2 mice containing woodchuck hepatocytes were infectable with woodchuck hepatitis virus (WHV) and showed WHV replication for at least 10 months with titers up to 1 ؋ 10 11 virions per ml in the peripheral blood. WHV-infected hepatocytes from chronic carrier woodchucks also established a persistent infection in uPA͞RAG-2 mice after an 8-to 12-week lag period of viremia. Although WHV envelope, core, and X proteins were produced in the uPA͞RAG-2 mice, no inflammatory host immune response was observed in the liver of WHV-replicating mice. A first antiviral test demonstrated a greater than four orders of magnitude drop in WHV titer in response to interferon ␣ treatment. WHV replication was upregulated by dexamethasone treatment. Comparison of precancerous lesions in donor woodchucks versus recipient uPA͞RAG-2 mice revealed an enrichment of dysplastic precancerous hepatocytes in transplanted mice. Clonal amplification of hepatocytes from a woodchuck with hepatocellular carcinomas was demonstrated by the detection of unique WHV DNA integration patterns in hepatocellular carcinomas that arose in uPA͞RAG-2 mice. In the absence of B or T cell-mediated immune responses, WHV establishes a persistent noncytotoxic infection of woodchuck hepatocytes in uPA͞RAG-2 chimeric mouse livers. Further studies of the kinetics of hepadnavirus infection and replication in quiescent and proliferating hepatocytes should increase our understanding of hepadnavirus spread and aid in the design of therapies to block or cure persistent infection.Hepatitis B virus (HBV) infection remains a major health problem with more than 350 million chronic HBV carriers worldwide, who are at risk for developing liver cirrhosis and hepatocellular carcinoma (HCC) (1-4). The development of effective therapies for eradicating HBV in chronic carriers has been limited by our incomplete understanding of mechanisms of viral persistence (5).Both humoral and cellular elements of the host immune response are important for HBV clearance. The humoral response to HBV antigens (i.e., antibodies to hepatitis B surface antigen) helps clear circulating virions and confers protection against reinfection, whereas T cell-mediated responses eliminate infected cells (6, 7). Recent work using HBV transgenic mice has shown that this process can be mediated by cytokines, such as tumor necrosis factor ␣ and interferon ␥. These cytokines have the capacity to down-regul...

Quantitative Gene Expression Analysis Reveals Transition of Fetal Liver Progenitor Cells to Mature Hepatocytes after Transplantation in uPA/RAG-2 Mice

“…In the heterozygous state some host cells can delete the transgene and form large regeneration nodules. 19,20 The transplanted EGFP-transgenic liver progenitor cells participated in tissue regeneration and formed cohesive cell clusters within the host liver of the uPA mice. The high frequency of small-and medium-sized EGFP-positive regeneration nodules in our experiments suggests efficient initial integration of fetal liver progenitor cells in the host liver and subsequent clonal expansion.…”

“…The uPA/ RAG-2 mice were generated by crossbreeding of uPAtransgenic mice originally described by Sandgren and colleagues 18 with the RAG-2 mouse as described elsewhere. 19,20 All animals were maintained and handled in accordance with institutional guidelines. For preparing fetal liver progenitor cells from EGFP embryos (embryonic day 13.5) the livers were removed under the binocular microscope.…”

Infection of human hepatocyte chimeric mouse with genetically engineered hepatitis B virus

“…A useful woodchuck hepatitis virus (WHV) infection model was established by Petersen et al 16 They showed highlevel replacement of uPA/Rag-2 knockout mice liver with woodchuck hepatocytes and development of high-level (1 ϫ 10 11 virion/mL) WHV viremia. Dandri et al 17 transplanted Tupaia hepatocyte into uPA/RAG-2 mice and showed up to 8.2 ϫ 10 7 genome equivalent/mL viremia.…”

Reduced Expression of DNA Damage Repair Genes High Mobility Group Box1 and Poly(ADP-ribose) Polymerase1 in Inactive Carriers of Hepatitis B Virus Infection-A Possible Stage of Viral Integration

“…Furthermore, integrations of linear double stranded HBV DNA (dsDNA) sequences in to host genome do occur in infected hepatocytes, particularly in the presence of DNA damage 5 and cell turnover. 6,7 The overlength pregenomic RNA (pgRNA) which act as a replication intermediate is reverse transcribed to form relaxed circular DNA (rcDNA), cccDNA as well as can produce truncated form of linear HBV dsDNA capable of integration. 8 As a consequence, persistence of cccDNA and integration of viral DNA in to host genome appeared as key factors responsible for relapse of HBV infection, failure of viral clearance after completion of antiviral therapy and development of HCC in CHB patients.…”