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Comparative Study
. 2021 Nov 4;10(11):3019.
doi: 10.3390/cells10113019.

Characterization of RNA Sensing Pathways in Hepatoma Cell Lines and Primary Human Hepatocytes

Wiebke Nicolay  1 , Rebecca Moeller  1   2 , Sina Kahl  1 , Florian W R Vondran  3   4 , Thomas Pietschmann  1 , Stefan Kunz  5 , Gisa Gerold  1   2   6   7
Affiliations
Comparative Study

Characterization of RNA Sensing Pathways in Hepatoma Cell Lines and Primary Human Hepatocytes

Wiebke Nicolay et al. Cells. .

Abstract

The liver is targeted by several human pathogenic RNA viruses for viral replication and dissemination; despite this, the extent of innate immune sensing of RNA viruses by human hepatocytes is insufficiently understood to date. In particular, for highly human tropic viruses such as hepatitis C virus, cell culture models are needed to study immune sensing. However, several human hepatoma cell lines have impaired RNA sensing pathways and fail to mimic innate immune responses in the human liver. Here we compare the RNA sensing properties of six human hepatoma cell lines, namely Huh-6, Huh-7, HepG2, HepG2-HFL, Hep3B, and HepaRG, with primary human hepatocytes. We show that primary liver cells sense RNA through retinoic acid-inducible gene I (RIG-I) like receptor (RLR) and Toll-like receptor 3 (TLR3) pathways. Of the tested cell lines, Hep3B cells most closely mimicked the RLR and TLR3 mediated sensing in primary hepatocytes. This was shown by the expression of RLRs and TLR3 as well as the expression and release of bioactive interferon in primary hepatocytes and Hep3B cells. Our work shows that Hep3B cells partially mimic RNA sensing in primary hepatocytes and thus can serve as in vitro model to study innate immunity to RNA viruses in hepatocytes.

Keywords: RIG-I; RNA virus; TLR3; arenavirus; coronavirus; hepatoma cells; innate immunity; interferon; liver; primary hepatocytes.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Transcript levels and protein levels of RNA sensors and adaptors in PHH and hepatoma cells. (a) RNA expression of RIG-I, MAVS, and MDA5 in indicated cell lines and in PHH derived from four different donors. RNA was extracted from whole-cell lysates, mRNA expression measured by qRT-PCR, normalized to GapDH mRNA expression, and plotted as 2−ΔCt values. Results are shown as mean ± SD of three independent experiments (with technical duplicates) and in the case of the PHH data were derived from one single experiment (n = 1, with technical duplicates). (b,d,f) Immunoblot for MAVS, RIG-I, and MDA5 in lysates from indicated cell lines. For detection of RIG-I and MDA5, cells were left untreated or pretreated with IFN-α (100 IU/mL). β-actin was used as a loading control. Results are representatives of three independent experiments. (c,e,g) Expression levels of the indicated proteins were quantified as immunoblot band density and shown relative to the β-actin loading control as mean ± SD of three independent experiments. One-way ANOVA, followed by Dunnett’s multiple comparison test *** p < 0.001, **** p < 0.0001.
Figure 2
Figure 2
Transcript levels and protein expression of TLRs and the adaptor molecule TRIF in PHH and hepatoma cells. RNA expression of TLR3, its adaptor TRIF, TLR7, and TLR8 (a) in indicated cell lines and in PHH derived from four different donors, as described in Figure 1a. Flow cytometric analysis of intracellular protein expression of TLR3 (b,c), TLR7 (d,e), and TLR8 (f,g) in indicated cell lines. Cells were permeabilized, stained against the respective TLR, an appropriate isotype control, or left untreated. Results are shown as one representative histogram (c,e,g) out of three independent experiments with 30,000 events per measurement for selective cell lines or depicted as MFI (b,d,f) as mean ± SD of three independent experiments. Jurkat cells served as a positive control. (h) Immunoblot for TRIF in lysates from indicated cell lines. β-actin was used as a loading control. Results are representatives of three independent experiments. (i) Expression levels of TRIF quantified as immunoblot band density and shown relative to the β-actin loading control as mean ± SD of three independent experiments. One-way ANOVA, followed by Dunnett’s multiple comparison test * p < 0.05, **** p < 0.0001.
Figure 3
Figure 3
Transcript level of IFNAR in PHH and hepatoma cells. RNA expression of IFNAR in tested cell lines and in PHH derived from four different donors. mRNA expression was measured by qRT-PCR as described in Figure 1a. One-way ANOVA, followed by Dunnett’s multiple comparison test **** p < 0.0001.
Figure 4
Figure 4
Induction of IFN-β expression after stimulation with indicated agonists of RNA sensors in PHH and hepatoma cells with and without IFN-α pre-stimulation. IFN-β RNA expression in PHHs and hepatoma cell lines mock-treated or pre-treated with IFN-α (100 IU/mL) followed by addition of TLR3 agonist poly(I:C) (1μg/mL) (a), TLR7/8 agonist R848 (1 μg/mL) (b) or transfected poly(I:C) (2.25 μg/well) (c) as RIG-I agonist. Transcript levels were measured by qRT-PCR 6 h after treatment for cell lines and 24 h after treatment for PHH, as described in Figure 1a. Data for the four different PHH donors are shown as mean ± SEM of single experiments performed in technical duplicates for each donor. For the cell lines, mean ± SEM of one experiment (performed in technical duplicates) is shown. Two-way ANOVA, followed by Dunnett’s multiple comparison test, **** p < 0.0001. Hepatoma cell lines were tested against PHH and only significant comparisons are indicated.
Figure 5
Figure 5
IFN-I secretion from PHH and hepatoma cell lines after stimulation with indicated RNA sensor agonists with and without IFN-α pre-stimulation (100 IU/mL) and further stimulated with TLR3 agonist poly(I:C) (1 μg/mL) (a), TLR7/8 agonist R848 (1 μg/mL), (b) or transfected poly(I:C) (2.25 μg/well), (c) as RIG-I agonist. Released IFN was measured by transferring cell cultures supernatants of stimulated cells to an IFN sensitive luciferase reporter cell line. U/mL was calculated using a recombinant IFN-I standard curve. Data for the four different PHH donors are shown as mean ±SEM of a single experiment performed in technical duplicates and for the cell lines as mean ± SEM of one representative of three independent experiments (performed in technical triplicates). Two-way ANOVA, followed by Dunnett’s multiple comparison test * p < 0.05, ** p < 0.01, **** p < 0.0001. n.d. = not detected.
Figure 6
Figure 6
Susceptibility and IFN response of PHH and hepatoma cells to CoV229E. (a) CoV infection in PHH and hepatoma cell lines. Cells were mock-treated or pretreated with IFN-α for 24 h and subsequently infected with CoV Renilla reporter virus (MOI 0.1) for 24 h. Infection with the reporter virus was determined 24 h post-infection by measuring luciferase activity. (b) IFN-I secretion from hepatoma cell lines and PHH (c) mock-treated or pretreated with IFN-α for 24 h followed by CoV infection. Released IFN was measured as described in Figure 5. Data for the five different PHH donors are shown as mean ±SD of a single experiment performed in technical duplicates and for the cell lines as mean ± SD of three independent experiments (performed in technical triplicates). Two-way ANOVA, followed by Dunnett’s multiple comparison test * p < 0.05, ** p < 0.01, **** p < 0.0001.
Figure 7
Figure 7
Susceptibility and IFN response of A549 and Hep3B cells to TCRV. Cells were mock-treated or pretreated with IFN-α for 24 h and subsequently infected with TCRV (MOI 0.01) for indicated time points (a). Viral titer was determined by titration on Vero cells and staining against the nucleoprotein. IFN-β mRNA expression was measured by qRT-PCR as described in Figure 1a (b). Results are given as a mean ± SEM of three independent experiments (with technical duplicates). Two-way ANOVA, followed by Turkeys’s multiple comparison test * p < 0.05, ** p < 0.01, *** p < 0.001.
Figure 8
Figure 8
Susceptibility of Hep3B miR-122 and Hep3B miR-122 subgenomic HCV replicon cells to TCRV (a) and JUNV Candid#1 (C#1) (b). Cells were infected with indicated arenaviruses at an MOI of 0.01 and supernatants were harvested at indicated time points. Viral titers were determined by titration on Vero cells and staining against the nucleoprotein. Results are given as a mean ±SEM of three (JUNV) or two (TCRV) independent experiments (with technical duplicates). Two-way ANOVA, followed by Sidak’s multiple comparison test ** p < 0.01, *** p < 0.001.
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References

    1. Protzer U., Maini M.K., Knolle P.A. Living in the liver: Hepatic infections. Nat. Rev. Immunol. 2012;12:201–213. doi: 10.1038/nri3169. - DOI - PubMed
    1. Gray K.K., Worthy M.N., Juelich T.L., Agar S.L., Poussard A., Ragland D., Freiberg A.N., Holbrook M.R. Chemotactic and inflammatory responses in the liver and brain are associated with pathogenesis of Rift Valley fever virus infection in the mouse. PLoS Negl. Trop. Dis. 2012;6:e1529. doi: 10.1371/journal.pntd.0001529. - DOI - PMC - PubMed
    1. Lukashevich I.S., Tikhonov I., Rodas J.D., Zapata J.C., Yang Y., Djavani M., Salvato M.S. Arenavirus-mediated liver pathology: Acute lymphocytic choriomeningitis virus infection of rhesus macaques is characterized by high-level interleukin-6 expression and hepatocyte proliferation. J. Virol. 2003;77:1727–1737. doi: 10.1128/JVI.77.3.1727-1737.2003. - DOI - PMC - PubMed
    1. Wang Y., Liu S., Liu H., Li W., Lin F., Jiang L., Li X., Xu P., Zhang L., Zhao L., et al. SARS-CoV-2 infection of the liver directly contributes to hepatic impairment in patients with COVID-19. J. Hepatol. 2020;73:807–816. doi: 10.1016/j.jhep.2020.05.002. - DOI - PMC - PubMed
    1. Sheahan T., Imanaka N., Marukian S., Dorner M., Liu P., PLoSs A., Rice C.M. Interferon lambda alleles predict innate antiviral immune responses and hepatitis C virus permissiveness. Cell Host Microbe. 2014;15:190–202. doi: 10.1016/j.chom.2014.01.007. - DOI - PMC - PubMed

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