Souphalone Luangsay1, Malika Ait-Goughoulte1, Maud Michelet2, Océane Floriot2, Marc Bonnin2, Marion Gruffaz2, Michel Rivoire3, Simon Fletcher4, Hassan Javanbakht4, Julie Lucifora2, Fabien Zoulim5, David Durantel6. 1. INSERM U1052, CNRS UMR_5286, Cancer Research Centre of Lyon (CRCL), 69008 Lyon, France; University of Lyon, Université Claude Bernard (UCBL), UMR_S1052, 69008 Lyon, France; Pharma Research & Early Development (pRED), Roche Innovation Center Basel, F. Hoffmann-La Roche AG, 4070 Basel, Switzerland. 2. INSERM U1052, CNRS UMR_5286, Cancer Research Centre of Lyon (CRCL), 69008 Lyon, France; University of Lyon, Université Claude Bernard (UCBL), UMR_S1052, 69008 Lyon, France. 3. Centre Léon Bérard (CLB), 69008 Lyon, France; INSERM U1032, 69003 Lyon, France. 4. Pharma Research & Early Development (pRED), Roche Innovation Center Basel, F. Hoffmann-La Roche AG, 4070 Basel, Switzerland. 5. INSERM U1052, CNRS UMR_5286, Cancer Research Centre of Lyon (CRCL), 69008 Lyon, France; University of Lyon, Université Claude Bernard (UCBL), UMR_S1052, 69008 Lyon, France; Hospices Civils de Lyon (HCL), 69002 Lyon, France; Institut Universitaire de France (IUF), 75005 Paris, France. Electronic address: fabien.zoulim@inserm.fr. 6. INSERM U1052, CNRS UMR_5286, Cancer Research Centre of Lyon (CRCL), 69008 Lyon, France; University of Lyon, Université Claude Bernard (UCBL), UMR_S1052, 69008 Lyon, France. Electronic address: david.durantel@inserm.fr.
Abstract
BACKGROUND & AIMS: HepaRG cells are considered as the best surrogate model to primary human hepatocyte (PHH) culture to investigate host-pathogen interactions. Yet their innate immune functions remain unknown. In this study, we explored the expression and functionality of Toll-like (TLR) and retinoic acid-inducible gene-1 (RIG-I)-like receptors (RLR) in these cells. METHODS: Gene and protein expression levels of TLR-1 to 9 and RLR in HepaRG were mainly compared to PHH, by RT-qPCR, FACS, and Western blotting. Their functionality was assessed, by measuring the induction of toll/rig-like themselves and several target innate gene expressions, as well as the secretion of IL-6, IP-10, and type I interferon (IFN), upon agonist stimulation. Their functionality was also shown by measuring the antiviral activity of some TLR/RLR agonists against hepatitis B virus (HBV) infection. RESULTS: The basal gene and protein expression profile of TLR/RLR in HepaRG cells was similar to PHH. Most receptors, except for TLR-7 and 9, were expressed as proteins and functionally active as shown by the induction of some innate genes, as well as by the secretion of IL-6 and IP-10, upon agonist stimulation. The highest levels of IL-6 and IP-10 secretion were obtained by TLR-2 and TLR-3 agonist stimulation respectively. The highest preventive anti-HBV activity was obtained following TLR-2, TLR-4 or RIG-I/MDA-5 stimulations, which correlated with their high capacity to produce both cytokines. CONCLUSIONS: Our results indicate that HepaRG cells express a similar pattern of functional TLR/RLR as compared to PHH, thus qualifying HepaRG cells as a surrogate model to study pathogen interactions within a hepatocyte innate system.
BACKGROUND & AIMS: HepaRG cells are considered as the best surrogate model to primary human hepatocyte (PHH) culture to investigate host-pathogen interactions. Yet their innate immune functions remain unknown. In this study, we explored the expression and functionality of Toll-like (TLR) and retinoic acid-inducible gene-1 (RIG-I)-like receptors (RLR) in these cells. METHODS: Gene and protein expression levels of TLR-1 to 9 and RLR in HepaRG were mainly compared to PHH, by RT-qPCR, FACS, and Western blotting. Their functionality was assessed, by measuring the induction of toll/rig-like themselves and several target innate gene expressions, as well as the secretion of IL-6, IP-10, and type I interferon (IFN), upon agonist stimulation. Their functionality was also shown by measuring the antiviral activity of some TLR/RLR agonists against hepatitis B virus (HBV) infection. RESULTS: The basal gene and protein expression profile of TLR/RLR in HepaRG cells was similar to PHH. Most receptors, except for TLR-7 and 9, were expressed as proteins and functionally active as shown by the induction of some innate genes, as well as by the secretion of IL-6 and IP-10, upon agonist stimulation. The highest levels of IL-6 and IP-10 secretion were obtained by TLR-2 and TLR-3 agonist stimulation respectively. The highest preventive anti-HBV activity was obtained following TLR-2, TLR-4 or RIG-I/MDA-5 stimulations, which correlated with their high capacity to produce both cytokines. CONCLUSIONS: Our results indicate that HepaRG cells express a similar pattern of functional TLR/RLR as compared to PHH, thus qualifying HepaRG cells as a surrogate model to study pathogen interactions within a hepatocyte innate system.
Authors: Marina Campos-Valdez; Hugo C Monroy-Ramírez; Juan Armendáriz-Borunda; Laura V Sánchez-Orozco Journal: Viruses Date: 2021-06-18 Impact factor: 5.048