Hepatitis B Virus Infection and Immunopathogenesis in a Humanized Mouse Model: Induction of Human-Specific Liver Fibrosis and M2-Like Macrophages

<div><p>The mechanisms of chronic HBV infection and immunopathogenesis are poorly understood due to a lack of a robust small animal model. Here we report the development of a humanized mouse model with both human immune system and human liver cells by reconstituting the immunodeficient A2/NSG (NOD.Cg-<i>Prkdc^scid Il2rg^tm1Wjl</i>/SzJ mice with human HLA-A2 transgene) with human hematopoietic stem cells and liver progenitor cells (A2/NSG-hu HSC/Hep mice). The A2/NSG-hu HSC/Hep mouse supported HBV infection and approximately 75% of HBV infected mice established persistent infection for at least 4 months. We detected human immune responses, albeit impaired in the liver, chronic liver inflammation and liver fibrosis in infected animals. An HBV neutralizing antibody efficiently inhibited HBV infection and associated liver diseases in humanized mice. In addition, we found that the HBV mediated liver disease was associated with high level of infiltrated human macrophages with M2-like activation phenotype. Importantly, similar M2-like macrophage accumulation was confirmed in chronic hepatitis B patients with liver diseases. Furthermore, gene expression analysis showed that induction of M2-like macrophage in the liver is associated with accelerated liver fibrosis and necrosis in patients with acute HBV-induced liver failure. Lastly, we demonstrate that HBV promotes M2-like activation in both M1 and M2 macrophages in cell culture studies. Our study demonstrates that the A2/NSG-hu HSC/Hep mouse model is valuable in studying HBV infection, human immune responses and associated liver diseases. Furthermore, results from this study suggest a critical role for macrophage polarization in hepatitis B virus-induced immune impairment and liver pathology.</p></div

HBV infection induces anti-HBV human immune response in A2/NSG/Fas-hu mice.

<p>HBV infection results in human immune response with induction of serum levels of human inflammatory cytokines (<b>A</b>), B cells response (serum IgM and IgG antibodies levels) (<b>B</b>) and T cell response (<b>C–F</b>). (<b>C–F</b>): Expansion of human T cells following stimulation with PHA or HBV antigens plus anti-CD28 mAb (14 days with IL7 and IL2) of human lymphoid tissue T cells from mock and HBV infected mice. Total human T cell expansion for PHA (<b>C</b>), HBV antigen and resulting percentage of expanded CD4+ and CD8+ T cells (<b>D</b>) following stimulation are presented. Error bars are shown as standard deviations. (<b>E</b>) HBV infection induced HLA-A2 restricted HBV-core (18–27)- or HBV-envelope (183–191)-specific CD8+ T cells. Antigen specific CD8+ T cells were detected after expansion with HBV antigens for 14 days as above. The immunodominant HBV-core (18–27) epitope induced higher levels of CD8+ T cells than the HBV-envelope (183–191) epitope. Spleen cells from mock-infected mice did not respond to stimulation with HBV antigens and showed no detectable antigen-specific T cells. (<b>F</b>) HBV infection and associated immune response induced liver infiltration of human T cells. Liver sections from Mock and HBV inoculated humanized mice sacrificed at 12–16 weeks post inoculation were stained with human CD3 (human T cells, red) antibodies. No significant leukocyte infiltration was observed in livers from mock animals.</p

Persistent HBV infection is associated with liver specific immune impairment.

<p>Expansion of human T cells following stimulation with PHA or HBV antigens plus anti-CD28 mAb (14 days with IL7 and IL2) of human T cells from liver or lymphoid tissues of mock, HBV plus neutralizing antibody or HBV infected mice. (<b>A</b>) Total human T cell expansion after 2 weeks of stimulation with HBV antigen was examined. (<b>B</b>) Th1 associated double positive cytokine production in PHA expanded T cells re-stimulated with PMA plus ionomycin was also examined. (<b>C</b>) Comparative analysis of HBsAg level in individual animals (Mock, PBS inoculated; HBV+, HBV inoculated with persistent HBV infection; HBV−, HBV inoculated with no infection; +NAb, HBV plus anti-HBsAg neutralizing antibody inoculated), and associated human liver and lymphoid tissue T cell expansion following HBV antigen stimulation. NA (Not applicable, indicating animals with low number of T cells in the liver below the assay requirements, thus not tested). ND (Not detectable).</p

HBV-induced liver disease and immune impairment is associated with M2-like macrophage activation.

<p>A2/NSG/Fas-hu HSC/Hep mice were infected with Mock or HBV and terminated at 12–16 weeks post infection. (<b>A</b>) Infiltrating monocyte/macrophage (CD68+, CD14^high, CD16^low/medium) from HBV-infected and control livers were stained for M1-like marker (CD86+) and M2-like markers (CD163+, CD206+). (<b>B</b>) Elevated levels of human M2-like macrophage gene expression profile in HBV infected humanized livers. (<b>C–D</b>) HBV-induced M2-like macrophage co-localized with human T cells (<b>C</b>), activated hepatic stellate cells and fibrotic regions (<b>D</b>) in infected humanized livers. (<b>E</b>) Sex and age matched control and chronic HBV patients at varying stages of liver diseases were examined to characterize macrophages (CD68+) in chronic HBV infection. Representative M2-like marker (CD206+) and fibrosis (Sirius red) were stained in healthy controls and chronic HBV-infected human livers. CHB, Chronic HBV infection; G1S1/G1S3, stage 1/stage 3; LC, chronic HBV associated hepatocyte cell carcinoma. Chronic HBV-induced fibrotic (CHB) and liver cancer (LC) patients exhibited elevated levels of macrophages (CD68+) of M2-like lineage (CD206+) in the liver. A black arrow denotes marking indicating same region.</p

HBV infection induces chronic hepatitis and human liver fibrosis.

<p>A2/NSG/Fas-hu HSC/Hep mice were inoculated with Mock or HBV and terminated at 12–16 weeks post infection. (<b>A</b>) Representative liver sections from sacrificed HBV infected or control mice stained with H&E and hCD45 to examine human leukocyte infiltration and Sirius red/fast green (SR/FG) and Masson's trichrome (MT) stains to examine liver fibrosis. Human specific α-SMA (alpha-smooth muscle actin) and GFAP (glial fibrillary acidic protein) antibodies were used to detect activated human hepatic stellate cell activation or myofibroblasts. Livers from two representative mice per group are shown. Gene expression analysis of human and mouse collagen 1 was examined using species-specific primers (<b>B</b>) and antibodies (<b>C</b>). A black arrow denotes marking indicating same region.</p

Anti-HBs neutralizing antibody prevents HBV infection and associated liver diseases.

<p>A2/NSG/Fas-hu HSC/Hep mice were inoculated with HBV +/− anti-HBs neutralizing antibody (NAb) terminated at 10–16 week post infection. (<b>A</b>) Serum level of HBs antigen was measured at sacrifice time point in mock, HBV alone or HBV + anti-HBs antibody groups. (<b>B</b>) Liver sections from mock, HBV or HBV plus anti-HBs antibody treated animals (representative two mice per group) were stained for HBV core or surface antigens. (<b>C</b>) Representative liver sections from mock, HBV or HBV plus anti-HBs antibody treated animals were stained with H&E and hCD45 to examine human leukocyte infiltration and Masson's trichrome (MT) stains to examine liver fibrosis. (<b>D</b>) Liver fibrosis was also examined using serum biomarkers (GGT and HA) in mock (n = 6), HBV (n = 7) or HBV plus anti-HBs antibody treated (n = 3) animals.</p

Persistent HBV infection in A2/NSG/Fas-hu HSC/Hep mice.

<p>(<b>A</b>) A2/NSG/Fas-hu mice or non-humanized mice were inoculated with PBS or HBV (1×10e3, 10e5 or 10e7 GE/mouse). Blood samples were collected at various times after infection. HBV genomic DNA was examined in sera from humanized mice infected with HBV at indicated titration dose and time points. (<b>B</b>) A2/NSG/Fas-hu mice or non-humanized mice were inoculated with PBS or HBV (1×10e6 GE/mouse). Blood samples were collected at various times after infection and HBs antigen in sera was measured by ELISA. (<b>C</b>) HBV genomic DNA was detected in sera from HBV-infected humanized mice at termination time points (14–16 wpi). †: Unable to bleed animal at later time points. (<b>D</b>) Liver samples were collected at termination time points (12–16 wpi). HBV core and surface antigens were detected in livers of HBV inoculated humanized mice and not in control groups.</p

Baseline characteristics of the patients treated with IFN/RBV.

*<p>Information of HCV genotype was unavailable for 17 patients and 4 patients who did not complete the treatment.</p

The association of rs12979860 with AST levels in chronic HCV patients.

<p>The AST levels were determined using a Synchron LX®20 autoanalyser. The AST levels were higher in patients carrying the T-allele. Data are mean±SD and representative of two experiments.</p

Characteristic of 896 study subjects.

a<p>HCV viral genotypes was avaliable for 283 individuals in the persistent group.</p>▴<p>P<0.05 and</p>#<p>P = 0.04 persistent vs clearence group;</p>#<p>P<0.05, persistent vs healthy controls;</p><p>Quantitative variables are displayed as mean and SD; HCV load are displayed as median and quartile range. ALT, AST, GGT, TG, TC (alanine aminotransferase, aspartate aminotransferase, glutamyl transpeptidase, triglycerides and total cholesterol).</p