Figure 1.
Murine-specific Fas antibody treatment promotes human liver reconstitution in A2/NSG-hu HSC/Hep mice.
A–B: Anti-mouse Fas (mFas) antibody enhances human hepatocyte repopulation in A2/NSG immunodeficient mice transplanted with human HSC and liver progenitor cells. Littermate A2/NSG mice transplanted with human CD34+ HSC and hepatocyte progenitors (HSC/Hep) were treated with vehicle or mFas activating antibody and sacrifice at 3 months post transplantation. (A) Liver sections from vehicle (PBS) or Fas antibody treated humanized mice were stained with anti-human HepPar1 monoclonal antibody. (B) % Hep Par1+ cells per total liver cells for each mouse was quantified using 5 different fields and summarized. *, p<0.05. (C) Elevated human serum albumin levels in Jo2 (mFas Ab) treated A2/NSG-hu HSC/Hep mice compared to pre-Jo2 (mFas Ab) treatment (n = 35, 3 cohorts). (D) Comparative analysis of human immune reconstitution (hCD45%) in A2/NSG/Fas-hu HSC/Hep mice and A2/NSG-hu HSC mice (n = 40, 3 cohorts). (E) Human immune reconstitution of liver and lymphoid tissue (spleen) in A2/NSG/Fas-hu HSC/Hep mice. Total leukocytes from indicated tissues were stained with human (hCD45+) and murine (mCD45+) leukocyte antibody plus dead cell marker (Y7).
Figure 2.
Persistent HBV infection in A2/NSG/Fas-hu HSC/Hep mice.
(A) 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) 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. (C) 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. (D) 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.
Figure 3.
HBV infection induces anti-HBV human immune response in A2/NSG/Fas-hu mice.
HBV infection results in human immune response with induction of serum levels of human inflammatory cytokines (A), B cells response (serum IgM and IgG antibodies levels) (B) and T cell response (C–F). (C–F): 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 (C), HBV antigen and resulting percentage of expanded CD4+ and CD8+ T cells (D) following stimulation are presented. Error bars are shown as standard deviations. (E) 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. (F) 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.
Figure 4.
HBV infection induces chronic hepatitis and human liver fibrosis.
A2/NSG/Fas-hu HSC/Hep mice were inoculated with Mock or HBV and terminated at 12–16 weeks post infection. (A) 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) and antibodies (C). A black arrow denotes marking indicating same region.
Figure 5.
Anti-HBs neutralizing antibody prevents HBV infection and associated liver diseases.
A2/NSG/Fas-hu HSC/Hep mice were inoculated with HBV +/− anti-HBs neutralizing antibody (NAb) terminated at 10–16 week post infection. (A) Serum level of HBs antigen was measured at sacrifice time point in mock, HBV alone or HBV + anti-HBs antibody groups. (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. (C) 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. (D) 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.
Figure 6.
Persistent HBV infection is associated with liver specific immune impairment.
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. (A) Total human T cell expansion after 2 weeks of stimulation with HBV antigen was examined. (B) Th1 associated double positive cytokine production in PHA expanded T cells re-stimulated with PMA plus ionomycin was also examined. (C) 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).
Figure 7.
HBV-induced liver disease and immune impairment is associated with M2-like macrophage activation.
A2/NSG/Fas-hu HSC/Hep mice were infected with Mock or HBV and terminated at 12–16 weeks post infection. (A) Infiltrating monocyte/macrophage (CD68+, CD14high, CD16low/medium) from HBV-infected and control livers were stained for M1-like marker (CD86+) and M2-like markers (CD163+, CD206+). (B) Elevated levels of human M2-like macrophage gene expression profile in HBV infected humanized livers. (C–D) HBV-induced M2-like macrophage co-localized with human T cells (C), activated hepatic stellate cells and fibrotic regions (D) in infected humanized livers. (E) 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.
Figure 8.
HBV induces M2-like activation in human macrophages.
(A–B) Polarized M1 or M2 monocyte-derived macrophages were treated with HBV (HepG2.2.15 derived), or mock (HepG2 derived) for 6 days and M1 and M2 macrophage activation was examined using (A) morphological analysis (oval/round shape – M1, spindle/fibroblast shape – M2) and (B) gene expression analysis (CD86 – M1, AMAC1 – M2). (C) Polarized or activated (enhanced polarization) M1 or M2 monocyte-derieved macrophages were treated with HBV (HepG2.2.15 derived), or mock (HepG2 derived) for 6 days and M1 and M2 macrophage activation was examined using cytokine analysis (IL12 – M1, IL10 – M2).