Single-cell atlas of the liver myeloid compartment before and after cure of chronic viral hepatitis.

BACKGROUND & AIMS: Chronic viral infections present serious public health challenges; however, direct-acting antivirals (DAAs) are now able to cure nearly all patients infected with hepatitis C virus (HCV), representing the only cure of a human chronic viral infection to date. DAAs provide a valuable opportunity to study immune pathways in the reversal of chronic immune failures in an in vivo human system. METHODS: To leverage this opportunity, we used plate-based single-cell RNA-seq to deeply profile myeloid cells from liver fine needle aspirates in patients with HCV before and after DAA treatment. We comprehensively characterised liver neutrophils, eosinophils, mast cells, conventional dendritic cells, plasmacytoid dendritic cells, classical monocytes, non-classical monocytes, and macrophages, and defined fine-grained subpopulations of several cell types. RESULTS: We discovered cell type-specific changes post-cure, including an increase in MCM7+STMN1+ proliferating CD1C+ conventional dendritic cells, which may support restoration from chronic exhaustion. We observed an expected downregulation of interferon-stimulated genes (ISGs) post-cure as well as an unexpected inverse relationship between pre-treatment viral load and post-cure ISG expression in each cell type, revealing a link between viral loads and sustained modifications of the host's immune system. We found an upregulation of PD-L1/L2 gene expression in ISG-high neutrophils and IDO1 expression in eosinophils, pinpointing cell subpopulations crucial for immune regulation. We identified three recurring gene programmes shared by multiple cell types, distilling core functions of the myeloid compartment. CONCLUSIONS: This comprehensive single-cell RNA-seq atlas of human liver myeloid cells in response to cure of chronic viral infections reveals principles of liver immunity and provides immunotherapeutic insights. CLINICAL TRIAL REGISTRATION: This study is registered at ClinicalTrials.gov (NCT02476617). IMPACT AND IMPLICATIONS: Chronic viral liver infections continue to be a major public health problem. Single-cell characterisation of liver immune cells during hepatitis C and post-cure provides unique insights into the architecture of liver immunity contributing to the resolution of the first curable chronic viral infection of humans. Multiple layers of innate immune regulation during chronic infections and persistent immune modifications after cure are revealed. Researchers and clinicians may leverage these findings to develop methods to optimise the post-cure environment for HCV and develop novel therapeutic approaches for other chronic viral infections.

Precision-Cut Liver Slices as an ex vivo model to evaluate antifibrotic therapies for liver fibrosis and cirrhosis.

Background: Precision-Cut Liver Slices (PCLS) are an ex vivo culture model developed to study hepatic drug metabolism. One of the main benefits of this model is that it retains the structure and cellular composition of the native liver. PCLS also represents a potential model system to study liver fibrosis in a setting that more closely approximates in vivo pathology than in vitro methods. The aim of this study was to assess whether responses to antifibrotic interventions can be detected and quantified with PCLS. Methods: PCLS of 250 µm thickness were prepared from four different murine fibrotic liver models: choline-deficient, L-amino acid-defined, high-fat diet (CDAHFD), thioacetamide (TAA), diethylnitrosamine (DEN), and carbon tetrachloride (CCl4). PCLS were treated with 5 µM Erlotinib for 72 hours. Histology and gene expression were then compared with in vivo murine experiments and TGF-ß1 activated hepatic stellate cells (HSCs). These types of PCLS characterization were also evaluated in PCLS from human cirrhotic liver. Results: PCLS viability in culture was stable for 72 hours. Treatment of erlotinib, an EGFR inhibitor significantly inhibited the expression of profibrogenic genes Il6, Col1a1 and Timp1 in PCLS from CDAHFD-induced cirrhotic mice, and Il6, Col1a1 and Tgfb1 in PCLS from TAA-induced cirrhotic rats. Erlotinib treatment of PCLS from DEN-induced cirrhotic rats inhibited the expression of Col1a1, Timp1, Tgfb1 and Il6, which was consistent with the impact of erlotinib on Col1a1 and Tgfb1 expression in in vivo DEN-induced cirrhosis. Erlotinib treatment of PCLS from CCl4-induced cirrhosis caused reduced expression of Timp1, Col1a1 and Tgfb1, which was consistent with the effect of erlotinib in in vivo CCl4-induced cirrhosis. In addition, in HSCs at PCLS from normal mice, TGF-ß1 treatment upregulated Acta2 (αSMA), while treatment with erlotinib inhibited the expression of Acta2. Similar expression results were observed in TGF-ß1 treated in vitro HSCs. Expression of MMPs and TIMPs, key regulators of fibrosis progression and regression, were also significantly altered under erlotinib treatment in PCLS. Expression changes under erlotinib treatment were also corroborated with PCLS from human cirrhosis samples. Conclusion: The responses to antifibrotic interventions can be detected and quantified with PCLS at the gene expression level. The antifibrotic effects of erlotinib are consistent between PCLS models of murine cirrhosis and those observed in vivo and in vitro. Similar effects were also reproduced in PCLS derived from patients with cirrhosis. PCLS is an excellent model to assess antifibrotic therapies that is aligned with the principles of Replacement, Reduction and Refinement (3Rs).