Characterization of pattern recognition receptor expression and functionality in liver primary cells and derived cell lines.

Different liver cell types are endowed with immunological properties, including cell-intrinsic innate immune functions that are important to initially control pathogen infections. However, a full landscape of expression and functionality of the innate immune signaling pathways in the major human liver cells is still missing. In order to comparatively characterize these pathways, we purified primary human hepatocytes, hepatic stellate cells, liver sinusoidal endothelial cells (LSEC), and Kupffer cells (KC) from human liver resections. We assessed mRNA and protein expression level of the major innate immune sensors, as well as checkpoint-inhibitor ligands in the purified cells, and found Toll-like receptors (TLR), RIG-I-like receptors, as well as several DNA cytosolic sensors to be expressed in the liver microenvironment. Amongst the cells tested, KC were shown to be most broadly active upon stimulation with PRR ligands emphasizing their predominant role in innate immune sensing the liver microenvironment. By KC immortalization, we generated a cell line that retained higher innate immune functionality as compared to THP1 cells, which are routinely used to study monocyte/macrophages functions. Our findings and the establishment of the KC line will help to understand immune mechanisms behind antiviral effects of TLR agonists or checkpoint inhibitors, which are in current preclinical or clinical development

HIF1α-mediated RelB/APOBEC3B downregulation allows Hepatitis B Virus persistence.

New therapeutic strategies against Hepatitis B virus (HBV) focus, among others, on the activation of the immune system to enable the infected host to eliminate HBV. Hypoxia inducible factor 1 alpha (HIF1α) stabilisation has been associated with impaired immune responses. HBV pathogenesis triggers chronic hepatitis-related scaring, leading inter alia to modulation of liver oxygenation and transient immune activation, both factors playing a role in HIF1α stabilisation. We addressed whether HIF1α interferes with immune-mediated induction of the cytidine deaminase APOBEC3B and subsequent covalently closed circular DNA (cccDNA) decay. Liver biopsies of chronic HBV patients (CHB) were analysed by IHC, and in situ hybridization. The effect of HIF1α induction/stabilisation on differentiated HepaRG or mice +/- HBV +/- LTβR-agonist (BS1) was assessed in vitro and in vivo. Induction of A3B and subsequent effects were analysed by RT-qPCR, immunoblotting, ChIP, ICC, and mass-spectrometry. Analysing CHB highlighted that areas with high HIF1α levels and low A3B expression correlated with high HBcAg, potentially representing a reservoir for HBV survival in immune-active patients. In vitro, HIF1α stabilisation, strongly impaired A3B expression and anti-HBV effect. Interestingly, HIF1α knock-down was sufficient to rescue the inhibition of A3B-upregulation and -mediated antiviral effects, whereas HIF2α knock-down had no effect. HIF1α stabilisation decreased the level of RelB protein but not its mRNA, which was confirmed in vivo. Noteworthy, this function of HIF1α was independent of its partner ARNT. In conclusion, inhibiting HIF1α expression or stabilisation represents a novel anti-HBV strategy in the context of immune-mediated A3B induction. High HIF1α, mediated by hypoxia or inflammation, offers a reservoir for HBV survival in vivo, and should be considered as a restricting factor in the development of novel immune therapies

Supplementary Material for: Characterization of Pattern Recognition Receptor Expression and Functionality in Liver Primary Cells and Derived Cell Lines

Different liver cell types are endowed with immunological properties, including cell-intrinsic innate immune functions that are important to initially control pathogen infections. However, a full landscape of expression and functionality of the innate immune signaling pathways in the major human liver cells is still missing. In order to comparatively characterize these pathways, we purified primary human hepatocytes, hepatic stellate cells, liver sinusoidal endothelial cells (LSEC), and Kupffer cells (KC) from human liver resections. We assessed mRNA and protein expression level of the major innate immune sensors, as well as checkpoint-inhibitor ligands in the purified cells, and found Toll-like receptors (TLR), RIG-I-like receptors, as well as several DNA cytosolic sensors to be expressed in the liver microenvironment. Amongst the cells tested, KC were shown to be most broadly active upon stimulation with PRR ligands emphasizing their predominant role in innate immune sensing the liver microenvironment. By KC immortalization, we generated a cell line that retained higher innate immune functionality as compared to THP1 cells, which are routinely used to study monocyte/macrophages functions. Our findings and the establishment of the KC line will help to understand immune mechanisms behind antiviral effects of TLR agonists or checkpoint inhibitors, which are in current preclinical or clinical development

A dual role for hepatocyte-intrinsic canonical NF-kappaB signaling in virus control.

peer reviewedBACKGROUND & AIMS: Hepatic innate immune control of viral infections has largely been attributed to Kupffer cells, the liver macrophages. However, also hepatocytes, the parenchymal cells of the liver, possess potent immunological functions in addition to their known metabolic functions. Owing to their abundance in the liver and known immunological functions, we aimed to investigate the direct anti-viral mechanisms employed by hepatocytes. METHODS: Using lymphocytic choriomeningitis virus (LCMV) as a model of liver infection, we first assessed the role of myeloid cells by depletion prior to infection. We investigated the role of hepatocyte-intrinsic innate immune signaling by infecting mice lacking canonical NF-kappaB signaling (IKKbeta(DeltaHep)) specifically in hepatocytes. In addition, mice lacking hepatocyte-specific interferon-alpha/beta signaling-(IFNAR(DeltaHep)), or interferon-alpha/beta signaling in myeloid cells-(IFNAR(DeltaMyel)) were infected. RESULTS: Here, we demonstrate that LCMV activates NF-kappaB signaling in hepatocytes. LCMV-triggered NF-kappaB activation in hepatocytes did not depend on Kupffer cells or TNFR1- but rather on TLR-signaling. LCMV-infected IKKbeta(DeltaHep) livers displayed strongly elevated viral titers due to LCMV accumulation within hepatocytes, reduced interferon-stimulated gene (ISG) expression, delayed intrahepatic immune cell influx and delayed intrahepatic LCMV-specific CD8(+) T-cell responses. Notably, viral clearance and ISG expression were also reduced in LCMV-infected primary hepatocytes lacking IKKbeta, demonstrating a hepatocyte-intrinsic effect. Similar to livers of IKKbeta(DeltaHep) mice, enhanced hepatocytic LCMV accumulation was observed in livers of IFNAR(DeltaHep), whereas IFNAR(DeltaMyel) mice were able to control LCMV-infection. Hepatocytic NF-kappaB signaling was also required for efficient ISG induction in HDV-infected dHepaRG cells and interferon-alpha/beta-mediated inhibition of HBV replication in vitro. CONCLUSIONS: Together, these data show that hepatocyte-intrinsic NF-kappaB is a vital amplifier of interferon-alpha/beta signaling pivotal for early, strong ISG responses, influx of immune cells and hepatic viral clearance

A dual role for hepatocyte-intrinsic canonical NF-κB signaling in virus control.

Using lymphocytic choriomeningitis virus (LCMV) as a model of liver infection, we first assessed the role of myeloid cells by depletion prior to infection. We investigated the role of hepatocyte-intrinsic innate immune signaling by infecting mice lacking canonical NF-κB signaling (IKKβΔHep) specifically in hepatocytes. In addition, mice lacking hepatocyte-specific interferon-α/β signaling-(IFNARΔHep), or interferon-α/β signaling in myeloid cells-(IFNARΔMyel) were infected