TLR8 agonist selgantolimod regulates Kupffer cell differentiation status and impairs HBV entry into hepatocytes via an IL-6-dependent mechanism.

OBJECTIVE: Achieving HBV cure will require novel combination therapies of direct-acting antivirals and immunomodulatory agents. In this context, the toll-like receptor 8 (TLR8) agonist selgantolimod (SLGN) has been investigated in preclinical models and clinical trials for chronic hepatitis B (CHB). However, little is known regarding its action on immune effectors within the liver. Our aim was to characterise the transcriptomic changes and intercellular communication events induced by SLGN in the hepatic microenvironment. DESIGN: We identified TLR8-expressing cell types in the human liver using publicly available single-cell RNA-seq data and established a method to isolate Kupffer cells (KCs). We characterised transcriptomic and cytokine KC profiles in response to SLGN. SLGN's indirect effect was evaluated by RNA-seq in hepatocytes treated with SLGN-conditioned media (CM) and quantification of HBV parameters following infection. Pathways mediating SLGN's effect were validated using transcriptomic data from HBV-infected patients. RESULTS: Hepatic TLR8 expression takes place in the myeloid compartment. SLGN treatment of KCs upregulated monocyte markers (eg, S100A12) and downregulated genes associated with the KC identity (eg, SPIC). Treatment of hepatocytes with SLGN-CM downregulated NTCP and impaired HBV entry. Cotreatment with an interleukin 6-neutralising antibody reverted the HBV entry inhibition. CONCLUSION: Our transcriptomic characterisation of SLGN sheds light into the programmes regulating KC activation. Furthermore, in addition to its previously described effect on established HBV infection and adaptive immunity, we show that SLGN impairs HBV entry. Altogether, SLGN may contribute through KCs to remodelling the intrahepatic immune microenvironment and may thus represent an important component of future combinations to cure HBV infection.

Hepatitis B Virus Targets Lipid Transport Pathways to Infect Hepatocytes.

BACKGROUND & AIMS: A single hepatitis B virus (HBV) particle is sufficient to establish chronic infection of the liver after intravenous injection, suggesting that the virus targets hepatocytes via a highly efficient transport pathway. We therefore investigated whether HBV uses a physiological liver-directed pathway that supports specific host-cell targeting in vivo. METHODS: We established the ex vivo perfusion of intact human liver tissue that recapitulates the liver physiology to investigate HBV liver targeting. This model allowed us to investigate virus-host cell interactions in a cellular microenvironment mimicking the in vivo situation. RESULTS: HBV was rapidly sequestered by liver macrophages within 1 hour after a virus pulse perfusion but was detected in hepatocytes only after 16 hours. We found that HBV associates with lipoproteins in serum and within machrophages. Electron and immunofluorescence microscopy corroborated a co-localization in recycling endosomes within peripheral and liver macrophages. Recycling endosomes accumulated HBV and cholesterol, followed by transport of HBV back to the cell surface along the cholesterol efflux pathway. To reach hepatocytes as final target cells, HBV was able to utilize the hepatocyte-directed cholesterol transport machinery of macrophages. CONCLUSIONS: Our results propose that by binding to liver targeted lipoproteins and using the reverse cholesterol transport pathway of macrophages, HBV hijacks the physiological lipid transport pathways to the liver to most efficiently reach its target organ. This may involve transinfection of liver macrophages and result in deposition of HBV in the perisinusoidal space from where HBV can bind its receptor on hepatocytes.

New insights into the early mechanisms of epileptogenesis in a zebrafish model of Dravet syndrome.

OBJECTIVE: To pinpoint the earliest cellular defects underlying seizure onset (epileptogenic period) during perinatal brain development in a new zebrafish model of Dravet syndrome (DS) and to investigate potential disease-modifying activity of the 5HT2 receptor agonist fenfluramine. METHODS: We used CRISPR/Cas9 mutagenesis to introduce a missense mutation, designed to perturb ion transport function in all channel isoforms, into scn1lab, the zebrafish orthologue of SCN1A (encoding voltage-gated sodium channel alpha subunit 1). We performed behavioral analysis and electroencephalographic recordings to measure convulsions and epileptiform discharges, followed by single-cell RNA-Seq, morphometric analysis of transgenic reporter-labeled γ-aminobutyric acidergic (GABAergic) neurons, and pharmacological profiling of mutant larvae. RESULTS: Homozygous mutant (scn1labmut/mut ) larvae displayed spontaneous seizures with interictal, preictal, and ictal discharges (mean = 7.5 per 20-minute recording; P < .0001; one-way analysis of variance). Drop-Seq analysis revealed a 2:1 shift in the ratio of glutamatergic to GABAergic neurons in scn1labmut/mut larval brains versus wild type (WT), with dynamic changes in neuronal, glial, and progenitor cell populations. To explore disease pathophysiology further, we quantified dendritic arborization in GABAergic neurons and observed a 40% reduction in arbor number compared to WT (P < .001; n = 15 mutant, n = 16 WT). We postulate that the significant reduction in inhibitory arbors causes an inhibitory to excitatory neurotransmitter imbalance that contributes to seizures and enhanced electrical brain activity in scn1labmut/mut larvae (high-frequency range), with subsequent GABAergic neuronal loss and astrogliosis. Chronic fenfluramine administration completely restored dendritic arbor numbers to normal in scn1labmut/mut larvae, whereas similar treatment with the benzodiazepine diazepam attenuated seizures, but was ineffective in restoring neuronal cytoarchitecture. BrdU labeling revealed cell overproliferation in scn1labmut/mut larval brains that were rescued by fenfluramine but not diazepam. SIGNIFICANCE: Our findings provide novel insights into early mechanisms of DS pathogenesis, describe dynamic cell population changes in the scn1labmut/mut brain, and present first-time evidence for potential disease modification by fenfluramine.