Abstract
Hepatocytes are key players in the innate immune response to liver pathogens but are challenging to study because of inaccessibility and a short half-life. Recent advances in in vitro differentiation of hepatocyte-like cells (HLCs) facilitated studies of hepatocyte–pathogen interactions. Here, we aimed to define the anti-viral innate immune potential of human HLCs with a focus on pattern recognition receptor (PRR)-expression and the presence of a metabolic switch. We analysed cytoplasmic PRR and endosomal toll-like receptor (TLR)-expression, as well as activity and adaptation of HLCs to an inflammatory environment. We found that transcript levels of retinoic acid inducible gene I (RIG-I), melanoma differentiation antigen 5 (MDA5), and TLR3 became downregulated during differentiation, indicating the acquisition of a more tolerogenic phenotype, as expected in healthy hepatocytes. HLCs responded to activation of RIG-I by producing interferons (IFNs) and IFN-stimulated genes. Despite low-level levels of TLR3, receptor expression was upregulated in an inflammatory environment. TLR3 signalling induced expression of proinflammatory cytokines at the gene level, indicating that several PRRs need to interact for successful innate immune activation. The inflammatory responsiveness of HLCs was accompanied by the downregulation of cytochrome P450 3A and 1A2 activity and decreased serum protein production, showing that the metabolic switch seen in primary hepatocytes during anti-viral responses is also present in HLCs.
Highlights
The liver has long been perceived exclusively as a metabolic organ where hepatocytes, the main cell type of the liver, are primarily responsible for nutrient metabolism, drug detoxification, and serum protein synthesis
Advances in in vitro differentiation of pluripotent stem cells (PSCs) into hepatocytes are transforming research in this area [3,4,5,6,7,8]. These models have already permitted more detailed studies of host–pathogen interactions, leading to new insights into the importance of hepatocytes in innate immune responses against viruses such as hepatitis C (HCV) and hepatitis B (HBV) [9,10,11,12]. These early studies showed advantages of induced pluripotent stem cells and human embryonic stem cells over hepatoma cell lines, which had classically been used for host–pathogen studies and which lack toll-like receptor 3 (TLR3) and show varying and often diminished retinoic acid inducible gene I (RIG-I) and type III interferon (IFN) responses [9,13,14,15]
After initial screening of pattern recognition receptor (PRR) expression in two induced pluripotent stem cells (iPSCs) and human embryonic stem cells (hESCs) lines, we focused our work on the iPSC-line P106 based on the finding that TLR3 expression was higher in iPSCs compared with hESCs and, more closely resembles TLR3 mRNA levels in primary human hepatocytes (PHHs)
Summary
The liver has long been perceived exclusively as a metabolic organ where hepatocytes, the main cell type of the liver, are primarily responsible for nutrient metabolism, drug detoxification, and serum protein synthesis. Hepatocytes are known to be important detectors of infection and inflammation, as well as major producers of acute phase proteins, complement components, and antimicrobial peptides They play critical roles in mediating systemic and local inflammatory and innate immune functions [1,2]. Advances in in vitro differentiation of pluripotent stem cells (PSCs) into hepatocytes are transforming research in this area [3,4,5,6,7,8] These models have already permitted more detailed studies of host–pathogen interactions, leading to new insights into the importance of hepatocytes in innate immune responses against viruses such as hepatitis C (HCV) and hepatitis B (HBV) [9,10,11,12]. While both Huh and Huh7.5 cells are important tools for studying the HCV-replication cycle in vitro, they are poor physiological models for the study of host defense mechanisms and the complex interplay between virus and host
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