Hepatitis B virus evades innate immunity of hepatocytes but activates cytokine production by macrophages

Abstract

Hepatitis B virus (HBV) infects hepatocytes specifically and causes immune-mediated liver damage. How HBV interacts with the innate immunity at the early phase of infection, either with hepatocytes or other cells in the liver, remains controversial. To address this question, we utilized various human cell-culture models and humanized Alb-uPA/SCID mice. All these models were unable to mount an interferon (IFN) response despite robust HBV replication. To elucidate the mechanisms involved in the lack of IFN response, we examined whether HBV actively inhibits innate immune functions of hepatocytes. By treating HBV-infected cells with known inducers of the IFN signaling pathway, we observed no alteration of either sensing or downstream IFN response by HBV. We showed that the DNA innate sensing pathways are poorly active in hepatocytes, consistent with muted innate immune recognition of HBV. Upon exposure to high-level HBV, human macrophages could be activated with increased inflammatory cytokine expressions.

Conclusion: HBV behaves like a "stealth" virus and is not sensed by, nor actively interferes with, the intrinsic innate immunity of infected hepatocytes. Macrophages are capable of sensing HBV, but require exposure to high HBV titers, potentially explaining the long "window period" during acute infection and HBV's propensity to chronic infection. (Hepatology 2017;66:1779-1793).

FIG. 1

In vitro and in vivo analysis of IFN response in hepatocyte against HBV. (A) In vitro cultured hepatocyte (HepG2-NTCP, dHepaRG, HLC, PHH) were either mock infected or infected with HBVcc (400 genomes/cell) for 7 days. Poly (I:C) transfection (2 µg/ml), SeV infection (1 HAU/ml) or HCV infection (0.5 TCID50/cell) were performed as positive control. 24 hours later, target genes expression were measured by qPCR. (B) HepG2-NTCP cells were either transfected with empty plasmid vector or plasmids containing 1.3-fold HBV genome of genotype A or genotype D. 48 hours later, target gene expression were determined by qPCR. Cells transfected with poly (I:C) (2 µg/ml) for 24 hours were served as positive control. Student unpaired two-tailed t tests, **P<0.01, ***P<0.001, n.s. = not significant. (C) Patient serum containing HBV (genotype A or C) or HBVcc (genotype D) were used to infect HLC for 7 days. HBV non-infected cells were transfected with poly (I:C) (1 µg/ml) for 24 hours to serve as positive control. (D and E) Alb-uPA/SCID mice were transplanted with PHHs from the same donor (noninfected mice: n = 3) and infected with HBV (genotype C, 10⁵ genomes) or HCV (genotype 1b, 10⁵ genomes). After virus infection, blood was sampled at indicated time points and mice were sacrificed either in the early stage of infection (10 d.p.i.) (HBV-infected: n = 5; HCV-infected: n = 5) or after the viremia reach the plateau (8 w.p.i.) (HBV-infected: n = 5; HCV-infected: n = 5). Viral titers in the blood were determined by q-PCR and hepatic IFNs expression were quantified by RT-qPCR with human specific Taqman probes and the detection limit were set as one. d.p.i. = day post infection. w.p.i. = week post infection. Mann-Whitney test, *P<0.05. For (A to C), means ± SD are shown. For (D and E), means ± SEM are shown.

FIG. 2

Analysis of IFN response of hepatocytes in the late stage of HBV infection. HepG2-NTCP cells were mock (dashed line) or HBV infected (400 genomes/cell, solid line) for 7 days and followed by poly (I:C) transfection (A), SeV infection (B), or poly (dA:dT) transfection (C) at dose indicated. 16 hours after transfection or 24 hours after SeV infection, cells were lysed and RT-qPCR was used to measure target genes expression. Similarly, 7 days post-HBV infection in HLC (D) and PHH (E), HBV-infected and mock-infected cells were treated in parallel with indicated dose of SeV or 2 µg/ml poly (dA:dT) as above. 7 days post-HBV infection in HepG2-NTCP (F) or PHH (G), cells were treated with IFN-α at indicated dose for 6 hours before gene expression analysis. Results are presented as fold induction. Means ± SD are shown. d.p.i. = day post infection.

FIG. 3

IRF3 and STAT1 nuclear translocation in HBV-infected HepG2-NTCP. HepG2-NTCP was either mock infected or HBV (400 genomes/cell) infected. On day 7 postinfection, cells were superinfected with SeV (100 HAU/ml) for 24 hours and followed by immune staining with antibodies against HBsAg in red and IRF3 (A) or STAT1 (B) in green. Yellow boxed areas are enlarged and shown below. White arrows indicate cells being double positive for HBsAg and nuclear translocation of IRF3 or STAT1. Scale bar = 20 µm.

FIG. 4

STAT1 nuclear translocation in IFN-α treated and HBV-infected hepatocytes. HepG2-NTCP (A) or PHH (B) were mock or HBV infected (400 genomes/cell) for 7 days and then treated with IFN-α (1000 IU/ml) for 1.5 hours. Cells were then fixed and stained with antibody against HBsAg (red) and STAT1 (green). Example cells showing STAT1 nuclear import are marked by white arrows. Scale bar = 20 µm.

FIG. 5

PRRs expression and functionality in cultured hepatocytes. Relative expression of RNA sensors (A) and DNA sensors (B) were determined in different hepatocyte models (relative to TBP) and the levels were compared to their expression in THP-macrophages, which were set as 100%. Experiment was performed in triplicates and means ± SD are shown. (C) Cultured cells were transfected with 2 µg/ml PRR specific ligands (described in Supporting Information) or mock transfected. IFNB, IFNL1 and ISG15 expression were determined by qPCR 16 hours posttreatment and results are presented as fold induction. Means ± SD are shown.

FIG. 6

Analysis of innate immune response in macrophages against HBV. (A) Primary human monocyte-derived macrophages were treated with indicated dose of HBV for 6 hours. Relative expression of target genes were measured by RT-qPCR and the results were expressed as fold change comparing to mock-treated cell except for IL6 which were compared to the detection limit. (B) HBV at 1 × 10⁸ genomes/mL or mock media prepared in parallel was applied to macrophage cultures for different lengths of time as indicated. Gene expression analysis was done as described above. (C) Macrophages derived from four independent donors were treated with 1 × 10⁸ copies/mL of HBV or similarly prepared mock or ETV-HBV (for ETV-HBV, data from one donor of the four are not available) for 6 hours. Gene expression analysis was performed similarly as mentioned above. (D) Macrophages were treated with 60 µM ETV or not for 6 hours, intracellular RNA were extracted to measure IL1B and TNFA expression. For (A), (B) and (D) means ± SD are shown. Student unpaired two-tailed t test is used. For (C) means ± SEM are shown and Mann-Whitney test is used. *P < 0.05, **P < 0.01, ****P < 0.0001, n.s. = not significant. n.d. = not detectable.