Modeling host interactions with hepatitis B virus using primary and induced pluripotent stem cell-derived hepatocellular systems

Abstract

Hepatitis B virus (HBV) chronically infects 400 million people worldwide and is a leading driver of end-stage liver disease and liver cancer. Research into the biology and treatment of HBV requires an in vitro cell-culture system that supports the infection of human hepatocytes, and accurately recapitulates virus-host interactions. Here, we report that micropatterned cocultures of primary human hepatocytes with stromal cells (MPCCs) reliably support productive HBV infection, and infection can be enhanced by blocking elements of the hepatocyte innate immune response associated with the induction of IFN-stimulated genes. MPCCs maintain prolonged, productive infection and represent a facile platform for studying virus-host interactions and for developing antiviral interventions. Hepatocytes obtained from different human donors vary dramatically in their permissiveness to HBV infection, suggesting that factors--such as divergence in genetic susceptibility to infection--may influence infection in vitro. To establish a complementary, renewable system on an isogenic background in which candidate genetics can be interrogated, we show that inducible pluripotent stem cells differentiated into hepatocyte-like cells (iHeps) support HBV infection that can also be enhanced by blocking interferon-stimulated gene induction. Notably, the emergence of the capacity to support HBV transcriptional activity and initial permissiveness for infection are marked by distinct stages of iHep differentiation, suggesting that infection of iHeps can be used both to study HBV, and conversely to assess the degree of iHep differentiation. Our work demonstrates the utility of these infectious systems for studying HBV biology and the virus' interactions with host hepatocyte genetics and physiology.

Keywords: HBV persistence; innate immunity; viral hepatitis.

Fig. 1.

HBV infection in micropatterned primary human hepatocytes is augmented by innate immune inhibition. (A) MPCC vs. RCC schematic. Hepatocytes in pink, fibroblasts in purple. (B) NTCP immunostaining: white circle marks hepatocyte island boundary. (Scale bar, 100 µm.) (C) NTCP Western blot. (D) NTCP quantitative RT-PCR (qRT-PCR; mean ± SEM, n = 3). (E) Schematic of viral life-cycle readouts used. (F, Left) ELISA for HBsAg, expressed as a mean ± SEM (n = 3), secreted into supernatant between 14 and 16 dpi; (Center) HBV 3.5-kb mRNA expression (one cell pellet per condition) at 16 dpi; (Right) Copies of cccDNA at 16 dpi, expressed as an average of biological duplicates ± range. (G) HBc immunostaining of MPCCs at the indicated days postinfection. Isotype-matched negative control shown. (Scale bars, 50 µm.) (H) Time course of HBV infection in MPCCs. (Left panels) HBsAg and HBeAg levels (average of triplicates) in supernatant; (Center) cccDNA levels; (Right panels) qRT-PCR for HBV 3.5-kb mRNA and total mRNA. Expression relative to DMSO-treated samples 7 dpi, one pellet per condition per experiment, verified across independent experiments. Dotted lines: limit of quantification (qPCR), cut-off (ELISA).

Fig. 2.

Temporal induction of ISGs in HBV infected MPCCs. Primary human hepatocyte MPCCs were either mock- or HBV-infected with concomitant JAKi or DMSO (vehicle control) treatment. RNA expression was analyzed for the indicated ISGs at 12 h, 24 h, 48 h, 72 h, 7 d, 11 d, and 16 d postinfection and reported relative to the mock-infected cells, expressed as a mean ± SEM (n = 3).

Fig. 3.

MPCCs as a platform for anti-HBV drug studies. (A) MPCCs treated with DMSO or JAKi, with or without entecavir or IFN-β, were incubated with HBV infectious serum for 24 h, followed by continued drug treatment every 2 d. Collected supernatants were analyzed for HBV DNA after 3 wk (Left), and for secreted HBsAg at the indicated time points (Right); results are expressed as a mean ± SEM, n = 3. (B) HBV-infected MPCCs treated with JAKi were dosed with either IFN-β or entecavir from 7 to 16 dpi, when cell pellets were analyzed for 3.5-kb mRNA expression relative to nonantiviral treated cells (one cell pellet per condition, verified across multiple experiments; Left) and for cccDNA, expressed as an average (per cell pellet) of duplicates ± range (Right). Also at 16 dpi, medium (last changed at 14 dpi) was analyzed for secreted HBV DNA, expressed as a mean ± SEM (n = 3) (Center). (C and D) JAKi or DMSO-treated MPCCs bearing primary human hepatocytes from different donors were incubated with HBV infectious serum and assayed at 16 dpi for HBsAg, expressed as average of duplicates, and cccDNA quantification, total copies per cell pellet. Dotted lines indicate limit of quantification (qPCR) or cut-off (ELISA).

Fig. 4.

HBV infection of iHeps is drug-sensitive and differentiation-dependent. (A–C) iPSCs were differentiated in a stepwise fashion, treated with JAKi and incubated with HBV infectious serum upon treatment at the indicated days of the differentiation protocol. (A) At 16 dpi, HBV 3.5-kb mRNA was quantified by qRT-PCR, shown relative to DMSO-treated cells infected at day 10 of differentiation, and expressed as the mean ± SEM across two separate experiments. (B) Also at 16 dpi, medium (last changed at 14 dpi) was analyzed for secreted HBsAg (mean ± SEM, n = 3). (C) Agarose gel separation of amplified cccDNA products (16 dpi) on a single gel with high- (++) and low- (+) expression positive controls (size mismatch because of slight curvature between distant lanes). (D) Differentiated iHeps (day 20 of differentiation) treated as indicated were incubated with HBV infectious serum, followed by HBsAg measurements in media, expressed as a mean ± SEM (n = 3). (E and F). iPSCs were incubated with HBV infectious serum at the indicated days of differentiation with DMSO or JAKi. (E) Southern blot of cellular DNA extracted at 16 dpi (SI Materials and Methods) using an HBV-specific probe; arrow indicates bands corresponding to HBV DNA (predicted to be relaxed, circular DNA at this size). (F) HBcAg immunofluorescent staining of DMSO- or JAKi- treated 16-dpi iPSCs infected at day 15 and 20 of differentiation; (Scale bar, 50 µm.) (G) ISG mRNA expression by qRT-PCR of HBV-infected iHeps at 16 dpi, normalized to the expression of HBV infected cells at day 7 of differentiation.