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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.

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The role of the endogenous interferon (IFN) system has been well characterized during IFN-based therapy for chronic hepatitis C virus (HCV) infection; less is known for direct-acting antivirals (DAAs). In this phase 3b open-label study, we assessed changes in IFN-stimulated genes (ISGs) in non-cirrhotic treatment-naïve or pegIFN/RBV-experienced HCV-GT1a-infected patients receiving paritaprevir/ritonavir/ombitasvir + dasabuvir + ribavirin (PrOD + R) for 12 weeks. ISG expression was quantified from peripheral blood mononuclear cells at baseline, treatment weeks (TW)2, TW4, TW8, end of treatment (EOT) and at post-treatment week 12. Paired sera were used to assess IFN-α/IFN-related chemokines/cytokines. Twenty-five patients were enrolled. Overall sustained virologic response (SVR)12 was 92% (no virologic failure [VF]) and 100% for those completing the study protocol. Two patients were excluded from the ISG analysis due to lack of post-treatment samples. The majority of ISGs were downregulated at TW2-TW4 (nadir TW4); however, a relative increase was observed at TW8-EOT, although levels were lower than baseline. This downregulation was accompanied by increases in IFN-α/IFN-related chemokines, a finding not observed with TH 1/2-related cytokines. Following SVR, ISG expression returned to TW2 levels. In conclusion, PrOD + R for 12 weeks was well-tolerated with no VF. Our data demonstrate dynamic alterations in innate immune profiles during highly potent IFN-free DAA therapy. The downregulation of ISG post-therapy suggests reversal of the "exhausted" ISG phenotype following SVR, and the rise in ISGs and IFN-α/IFN-responsive chemokines late during therapy suggests resetting of IFN responsiveness that may be relevant in determining duration of or immunological sequelae from DAA therapy, including HBV reactivation.

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The biological factors that promote inflammation or nonalcoholic steatohepatitis (NASH) in the setting of nonalcoholic fatty liver disease remain incompletely understood. Clinical studies have demonstrated an association between obstructive sleep apnea (OSA) and both inflammation and fibrosis in NASH, but the mechanism has not been identified. In this study, we use in vitro modeling to examine the impact of intermittent hypoxia on the liver. Hepatocyte, stellate cell, and macrophage cell lines were exposed to intermittent or sustained hypoxia. Candidate genes associated with inflammation, fibrosis, and lipogenesis were analyzed. Circulating cytokines were assessed in human serum of patients with nonalcoholic fatty liver disease. Intermittent hypoxia results in significant induction of interleukin (IL)-6 expression in both hepatocytes and macrophages. The increase in IL-6 expression was independent of hypoxia inducible factor 1 induction but appeared to be in part related to antioxidant response element and nuclear factor kappa B activation. Mature microRNA 365 (miR-365) has been demonstrated to regulate IL-6 expression, and we found that miR-365 expression was decreased in the setting of intermittent hypoxia. Furthermore, macrophage cell lines showed polarization to an M1 but not M2 phenotype. Finally, we found a trend toward higher circulating levels of IL-6 in patients with OSA and NASH. Conclusion: Intermittent hypoxia acts as a potent proinflammatory stimulus, resulting in IL-6 induction and M1 macrophage polarization. Increased IL-6 expression may be due to both induction of antioxidant response element and nuclear factor kappa B as well as inhibition of miR-365 expression. Higher levels of IL-6 were observed in human samples of patients with OSA and NASH. These findings provide biological insight into mechanisms by which obstructive sleep apnea potentiates inflammation and fibrosis in patients with fatty liver disease. (Hepatology Communications 2017;1:326-337).

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The first line of defense against viral infection is the interferon (IFN) response, which culminates in the expression of hundreds of proteins with presumed antiviral activity, and must be overcome by a virus for successful replication. The nonstructural NSs protein is the primary IFN antagonist encoded by Bunyamwera virus (BUNV), the prototype of the Orthobunyavirus genus and the family Bunyaviridae. The NSs protein interferes with RNA polymerase II-mediated transcription, thereby inhibiting cellular mRNA production, including IFN mRNAs. A recombinant virus, rBUNdelNSs, that is unable to express the NSs protein does not inhibit cellular transcription and is a strong IFN inducer. We report here that cells stimulated into the antiviral state by IFN-ß treatment were protected against wild-type BUNV and rBUNdelNSs infection but addition of IFN-ß after infection had little effect on the replication cycle of either virus. By screening a panel of cell lines that overexpressed individual IFN-stimulated genes, we found that protein kinase R (PKR), MTAP44, and particularly viperin appreciably restricted BUNV replication. The enzymatic activities of PKR and viperin were required for their inhibitory activities. Taken together, our data show that the restriction of BUNV replication mediated by IFN is an accumulated effect of at least three IFN-stimulated genes that probably act on different stages of the viral replication cycle.

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