Volume 140, Issue 7, Pages 2074-2083.e2 (June 2011) Hepatitis B Virus Limits Response of Human Hepatocytes to Interferon-α in Chimeric Mice  Marc Lütgehetmann, Till Bornscheuer, Tassilo Volz, Lena Allweiss, Jan–Hendrick Bockmann, Joerg M. Pollok, Ansgar W. Lohse, Joerg Petersen, Maura Dandri  Gastroenterology  Volume 140, Issue 7, Pages 2074-2083.e2 (June 2011) DOI: 10.1053/j.gastro.2011.02.057 Copyright © 2011 AGA Institute Terms and Conditions

Figure 1 Kinetics of IFN-α–induced activation of ISGs in human hepatocytes within the liver of uPA/SCID mice. Human chimeric mice were sacrificed either before (0 hours) or at different time points (as indicated) after IFN injection to determine steady-state levels of human (top panel) MxA, (middle panel) OAS-1, and (bottom panel) HLA-A RNAs by real-time polymerase chain reaction and using human specific primers. ISG expression levels are expressed on a log scale and are normalized to human glyceraldehyde-3-phosphate dehydrogenase (GAPDH) amounts. Maximal activation was achieved 12 hours postinjection and in most cases returned to baseline within 24 hours. Gastroenterology 2011 140, 2074-2083.e2DOI: (10.1053/j.gastro.2011.02.057) Copyright © 2011 AGA Institute Terms and Conditions

Figure 2 Temporary inhibition of HBV replication induced by daily IFN-α treatment in human chimeric mice. Twenty HBV-infected mice displaying median viremia of 7 × 107 HBV copies/mL serum were treated either with IFN-α (n = 12) or saline (n = 8). After 5 days of treatment, mice were sacrificed at 8 hours (n = 8), 12 hours (n = 5), and 24 hours (n = 3) after the last IFN injection to determine viremia reduction (A), intrahepatic covalently closed circular DNA (cccDNA) copies per human hepatocytes (B), as well as reduction of virion productivity. Between 8 and 12 hours post-IFN administration, a significant, but short lasting, decrease of both (C) pgRNA/cell and (D) rcDNA/cccDNA molecule was determined. Gastroenterology 2011 140, 2074-2083.e2DOI: (10.1053/j.gastro.2011.02.057) Copyright © 2011 AGA Institute Terms and Conditions

Figure 3 Impaired responsiveness of human ISGs to IFN-α treatment in HBV chronically infected livers of human chimeric mice. Intrahepatic RNA expression levels of (A) human MxA and OAS-1, as well as of (B) TLR2, TLR4, and adaptor molecule MyD88 were analyzed at 8 hours or 12 hours after the last IFN-injection both in HBV-infected (n = 16) and uninfected control mice (n = 18) and compared to levels found in the livers of untreated HBV-infected (n = 8) and uninfected (n = 7) mice. *Enhancement of ISG expression levels was highly significant (P < .001) in uninfected animals, although it did not reach significance in HBV-chronically infected mice (n.s.). Gastroenterology 2011 140, 2074-2083.e2DOI: (10.1053/j.gastro.2011.02.057) Copyright © 2011 AGA Institute Terms and Conditions

Figure 4 IFN-mediated up-regulation of a subset of genes of the antigen presentation is impaired by HBV infection in vivo. Quantitative intrahepatic RNA measurements performed in livers of animals sacrificed either at 8 and 12 hours post-IFN injection revealed that IFN-induced enhancement of (A) HLA-A and (B) HLA-E expression levels was not significantly restrained by HBV infection, while induction of (C) human TAP-1 gene expression, a key regulatory element of the antigen presentation pathway, reached significance only in mice harboring uninfected human hepatocytes. *P < .001. Gastroenterology 2011 140, 2074-2083.e2DOI: (10.1053/j.gastro.2011.02.057) Copyright © 2011 AGA Institute Terms and Conditions

Figure 5 HBV infection blocks the IFN-induced enhancement of TAP-1, but not of HLA proteins in human hepatocytes repopulating the livers of uPA/ SCID mice. Confocal analysis of immunostained liver samples shows (A) barely detectable levels of HLA class I proteins (green) in uninfected and untreated human hepatocytes (left, upper panel), which are slightly elevated in untreated, HBV-positive mouse livers (left, lower panel) and become strongly increased after IFN-α treatment both in uninfected and HBV-infected human hepatocytes (right panels). (B) Double staining with human specific cytokeratin 18 antibodies (red, left panels) permits identification of human hepatocytes in uPA mouse livers as well as protein levels of human TAP-1 (green, right panels). In agreement with the RNA measurements, IFN treatment specifically increases human TAP-1 protein levels in the liver of uninfected animals (upper panel), while TAP-1 levels remain barely detectable in HBV-positive mice. Gastroenterology 2011 140, 2074-2083.e2DOI: (10.1053/j.gastro.2011.02.057) Copyright © 2011 AGA Institute Terms and Conditions

Figure 6 The nuclear translocation of STAT-1 in response to IFN-α is inhibited in HBV-infected human hepatocytes. Immunofluorescence analysis of uninfected (upper panels) and HBV-infected (lower panels) chimeric livers derived from mice sacrificed 4 hours after injection of IFN-α. Double staining with human SP100 antibodies (red-pink dots in cell nuclei, left panels) and STAT antibodies (green, right panels) permit to identify the human hepatocytes within the mouse livers, as well as nuclear accumulation of STAT-1 in not-infected human hepatocytes. 4′,6-Diamidino-2-phenylindole staining (left panels) was also used to identify all cell nuclei in liver sections. Gastroenterology 2011 140, 2074-2083.e2DOI: (10.1053/j.gastro.2011.02.057) Copyright © 2011 AGA Institute Terms and Conditions

Figure 7 Suppression of HBV replication induced by ETV treatment was unable to re-establish the IFN-α response in HBV-infected hepatocytes. Human specific primers were used to determine expression levels of the indicated ISGs both in uninfected (light gray columns), HBV-infected (dark gray columns), and ETV-treated mice (black columns). Shown are the folds of ISGs induction relative to values determined in the respective untreated control groups (either uninfected or HBV-infected mouse livers). Gastroenterology 2011 140, 2074-2083.e2DOI: (10.1053/j.gastro.2011.02.057) Copyright © 2011 AGA Institute Terms and Conditions

Supplementary Figure 1 Expression levels of human and murine ISGs were quantitated by real-time polymerase chain reaction before and after administration of human IFN-α. Human and mouse glyceraldehyde-3-phosphate dehydrogenase (GAPDH) housekeeping genes were used for normalization. (Top panel) Log expression of the indicated human ISGs was determined using human specific primers not cross-reacting with murine sequences. The specificity of the designed primers was validated on complementary DNA samples derived from not-transplanted mouse liver, human-chimeric mouse liver, and human liver tissue. (Bottom panel) Primers recognizing ISG murine transcripts showed that injection of human IFN-α (1350 IU/g body weight human) failed to induce induction of the ISGs analyzed in murine liver cells, because transcription levels remained comparable to levels determined in untreated control littermates. Gastroenterology 2011 140, 2074-2083.e2DOI: (10.1053/j.gastro.2011.02.057) Copyright © 2011 AGA Institute Terms and Conditions

Supplementary Figure 2 Changes in levels of viremia (Log HBV-DNA copies/mL serum) were determined in the same HBV-infected mice before treatment (baseline [BL]) and at different time points after IFN-α administration. Viremia levels measured in untreated control mice used for the intrahepatic measurements are also shown (control group). Gastroenterology 2011 140, 2074-2083.e2DOI: (10.1053/j.gastro.2011.02.057) Copyright © 2011 AGA Institute Terms and Conditions

Supplementary Figure 3 Intracellular amounts of HBV proteins do not differ between untreated and ETV + IFN-α–treated mice. HBcAg staining (green, upper panels) of liver tissues obtained from HBV-infected chimeric mice receiving ETV for 2 weeks followed by 5 days of combination therapy with IFN-α + ETV (right upper panel) was comparable to the staining obtained from untreated HBV-infected chimeric livers (left upper panel). As expected, polymerase inhibitors strongly reduced viremia levels but not intracellular production of HBV proteins. Double staining with human cytokeratin 18 (CK-18) antibodies permitted to identify human cells in uPA/SCID mouse livers (lower panels). Gastroenterology 2011 140, 2074-2083.e2DOI: (10.1053/j.gastro.2011.02.057) Copyright © 2011 AGA Institute Terms and Conditions