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 uPA/RAG-2 mouse liver. uPA/RAG-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 x 10(11) virions per ml in the peripheral blood. WHV-infected hepatocytes from chronic carrier woodchucks also established a persistent infection in uPA/RAG-2 mice after an 8- to 12-week lag period of viremia. Although WHV envelope, core, and X proteins were produced in the uPA/RAG-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 alpha treatment. WHV replication was up-regulated by dexamethasone treatment. Comparison of precancerous lesions in donor woodchucks versus recipient uPA/RAG-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 uPA/RAG-2 mice. In the absence of B or T cell-mediated immune responses, WHV establishes a persistent noncytotoxic infection of woodchuck hepatocytes in uPA/RAG-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.

Figure 1

Migration pattern of mouse (lane 1) and woodchuck (lane 2) serum albumin in a Coomassie blue-stained gel. The migration pattern of serum albumin from a representative uPA/RAG-2 mouse containing woodchuck hepatocytes shows both mouse and woodchuck serum albumin (lane 3).

Figure 2

Woodchuck DNA and WHx, WHc, and WHs proteins in uPA/RAG-2 mice transplanted with WHV-positive woodchuck hepatocytes. (A) A Southern blot of genomic DNAs, hybridized with a woodchuck genome DNA probe. Lanes 1–4 present mixtures of genomic woodchuck liver DNA and untransplanted uPA/RAG-2 mouse genomic liver DNA with signals reflecting 100%, 50%, 20%, and 1%, woodchuck hepatocyte DNA, respectively. Lane 5 presents 100% untransplanted uPA/RAG-2 mouse DNA. Woodchuck DNA is undetectable in the spleen of transplanted uPA/RAG-2 mice (lane 6) but present in various amounts in all lobes of the liver of the uPA/RAG-2 mouse transplanted with WHV-positive woodchuck hepatocytes (lanes 7–11). (B) WHV DNA forms, detectable in uPA/RAG-2 mouse genomic liver DNA, hybridized with a WHV DNA probe. Lanes: 1, uPA/RAG-2 mouse liver; 2, uPA/RAG-2 mouse liver transplanted with WHV-positive woodchuck hepatocytes; 3, donor woodchuck genomic liver DNA. OC, open circular DNA; RF, replicative WHV DNA forms; CCC, covalently closed circular DNA; SS, single-stranded DNA. Lane M contains molecular size markers. (C) Immunoprecipitates with WHx antiserum from uPA/RAG-2 mouse liver (lane 1), from uPA/RAG-2 mouse transplanted with WHV-positive woodchuck hepatocytes (lane 2), and from donor woodchuck liver (lane 3). (D) Immunoblotting with WHc antiserum of hepatocyte extracts from uPA/RAG-2 mouse (lane 1), from uPA/RAG-2 mouse transplanted with WHV-positive woodchuck hepatocytes (lane 2), and from donor woodchuck (lane 3). (E) WHs proteins in uPA/RAG-2 mice serum. Immunoblotting with WHs antiserum of uPA/RAG-2 mouse 249 and uPA/RAG-2 mouse sera transplanted with WHV-positive woodchuck hepatocytes (uPA/RAG-2 mice 496, 969, 1063, and 1418). Lane WC was probed with WHV-positive woodchuck serum.

Figure 3

(A) Detection of WHcAg in a uPA/RAG-2 mouse liver containing WHV-positive woodchuck hepatocytes by immunostaining with a WHc antiserum. A nodule containing transplanted WHV-positive woodchuck hepatocytes (lighter area, rhodamine light) and host mouse hepatocytes that presumably deleted the uPA transgene (darker stained area). (×200.) (B) DPPIV staining of bile canaliculi in uPA/RAG-2 mouse liver containing woodchuck hepatocytes. (×200.) Bile canaliculi are visible between mouse hepatocytes (darker staining) and transplanted woodchuck hepatocytes (lighter staining). Nuclei are counterstained with hematoxylin.

Figure 4

Effect of interferon α and dexamethasone upon WHV secretion in uPA/RAG-2 mice. Each trace shows data from individual uPA/RAG-2 mice containing WHV-secreting woodchuck hepatocytes. Arrows mark starting point and withdrawal of agents. Time points mark the collection of serum samples. The dashed line represents the threshold of sensitivity for the dot blot analysis.

Figure 5

Hematoxylin/eosin staining of frozen liver sections from donor woodchucks (A, C, E, and G) and uPA/RAG-2 mice recipients (B, D, F, and H) after transplantation of WHV-positive woodchuck hepatocytes. A woodchuck liver (no. 2765) contained altered hepatic foci (A, ×200; C, ×1,000; right side of arrows marks border), with large nuclei and prominent nucleoli that appear similar to transplanted woodchuck hepatocytes in uPA/RAG-2 mouse liver (B, ×200; D, ×1,000; right side of arrows marks border). (E and G) Examples of an HCC and cholangiocarcinoma, respectively, from a woodchuck (no. 4940) chronically infected with WHV. (×200.) (F and H) Presence of an HCC (F) and a cholangiocarcinoma (H) in a uPA/RAG-2 mouse after transplantation of hepatocytes from the donor woodchuck shown in E and G. (×200.)

Figure 6

(A) Southern blot analysis of uPA/RAG-2 mouse liver genomic DNA with a woodchuck genomic DNA probe (lanes 3 and 4). Woodchuck DNA is detectable in tumors arising in uPA/RAG-2 mice (lane 3). Lane 4 is a negative control. Unique WHV DNA integrations in DNA from the tumor of lane 3 are detected with a WHV DNA probe (lane 2, with lane 1 as negative control) that are different from WHV DNA integrations in donor woodchuck tumor DNA samples (B, lanes 1–3).