Chronic HCV infection induces interferon and dysregulates immune responses through inflammation and chronic antigenic stimulation. Antiviral drugs can cure HCV, providing a unique opportunity to examine the immunological restoration that does and does not occur when a chronic viral infection is eradicated. We quantified blood cytokines levels and used mass cytometry to immunophenotype peripheral blood mononuclear cells before and after HCV cure in 2 groups of patients and controls. At baseline, serum interferon α and soluble CD163 (a macrophage product) were elevated in both liver transplant and nonliver transplant patients compared to controls; the frequencies of several peripheral blood mononuclear cell populations differed from controls; and programmed death protein 1-positivity was increased in nearly all T cell subsets. Many abnormalities persisted after HCV cure, including elevated programmed death protein 1 expression on CD4 naïve and central memory T cells, elevated soluble CD163, and expansion of the plasmablast/plasma cell compartment. Several myeloid-lineage subsets, including Ag-presenting dendritic cells, remained dysregulated. In mechanistic studies, interferon α treatment increased programmed death protein 1 on human T cells and increased T cell receptor signaling. The data identify immunological abnormalities that persist after curative HCV treatment. Before cure, high levels of interferon α may stimulate programmed death protein 1 expression on human T cells, causing persistent functional changes.

Dear Editor, We used publicly available single-cell RNA sequencing data to identify conserved tissue macrophage populations in human organs. Among the subsets, we found a rare population of metallothionein-expressing macrophages that are present in all vital organs analyzed. We deeply phenotype this subset and determine their localization in the human liver. In the first phase, we collected data from 10 livers,1 21 kidneys2 and 60 lungs,3 and selected myeloid-lineage cells using published annotations and lineage markers: CD68, S100, HLA-II, LYZ, C1Q, and CD74 (Figure S1). After removing dendritic cells, we subclustered monocytes and macrophages (liver, 8,197; kidney, 5,005; lung, 121,536), and identified eight subclusters in the monocyte-macrophage (mono-mac) space, defined by their differentially expressed genes (DEGs) (Figure 1A and Figure S2). We evaluated the relatedness of subclusters across organs by determining the Pearson correlations of fold-changes of 1126 DEGs that were significant [p.adj < .05 (Bonferroni)] in at least two organs (Figure 1B). Cross-organ correlations confirmed the relatedness of most subsets and were highest for monocytes and cycling macrophages. A survey of the mono-mac subtypes present in at least two human organs revealed that the abundances of most were similar across organs, but MAC_RES was disproportionately large in the lung (alveolar macrophages) and absent from the kidney (Figure 1C). Subset abundances were compared between fibrotic and control organs and differed significantly for three: monocytes, MAC_RES and SPP1+ macrophages (Figure S2). While most of the eight subsets are widely known, we found an unexpected, small population of macrophages (∼2.5% of each organ) that highly expressed genes coding for metallothioneins (MAC_MT). Metallothioneins are low molecular weight, cysteine-rich proteins that bind metal ions. They enhance angiogenesis, protect against oxidative stress and metal toxicity, and modulate macrophage function.4 Gene expression patterns of MAC_MTs were highly correlated across all three organs. Using a Wilcoxon rank-sum test to calculate DEGs, nine genes were significant [FC > 0, p.adj < .05 (Bonferroni)] in MAC_MTs in all three organs (Figure 1D). Genes encoding metallothioneins were highly expressed by MAC_MTs only, unlike CTSL and SGK1, which were expressed by multiple sub-clusters. Of interest, MTF1 and MTF2, which encode metallothionein transcription factors, were minimally expressed in MAC_MTs but were highly expressed in the CYCLING subset, which may provide a local source of MAC_MTs. In each organ, two gene signatures were developed, one distinguished MAC_MTs from other non-dendritic myeloid-lineage cells, and the other distinguished MAC_MTs from all other cells in the organ. To generate each signature, we selected DEGs [FC > 0.75, p.adj < .05 (Bonferroni)] whose expression was significantly greater in MAC_MTs than in the second-highest subset [FC > 0, p.adj < .05 (Bonferroni)]. This method generates gene signatures that can identify a subset of interest against a background of other cells. It removes genes that are highly expressed in other cell types and goes beyond an arbitrary number of top DEGs. We next scored all individual cells based on their expression of both MAC_MT organ-specific gene signatures and compared scores across the subsets (Figure 1E and Table S1). The signatures of MAC_MTs vs. myeloid-lineage cells were dominated by MT1 genes and MT2A but also included other immune response and metabolism-associated genes. The signature of liver MAC_MTs included HAMP (encodes hepcidin), an iron regulator. The signatures distinguished MAC_MTs from other myeloid-lineage cells, as demonstrated by UMAP overlays (Figure S3). The whole-organ signatures distinguished MAC_MTs of the liver and lung from all other cell types in the source organ (Figure S4), but did not distinguish kidney MAC_MTs with confidence, likely due to the high expression of metallothionein-encoding genes in kidney epithelial cells (Figure S5). To infer the differentiation status of MAC_MTs, we performed pseudotemporal analysis with monocytes as the starting node. The trajectories suggest that MAC_MTs originate from monocytes and are not terminally differentiated (Figure 1F). Lineage tracing studies are needed to further define their origin and progeny. To predict the potential functional roles of MAC_MTs in tissue, we inferred ligand-receptor interactions between MAC_MTs and all other cell types, focusing on ligands secreted by MAC_MTs. We identified 15 interactions conserved across tissues (Figure S6). Gene set enrichment analysis highlighted biological processes involving cellular migration, vascularization and angiogenesis. The genes driving these enrichments (GRN, TNFSF12, IL1B, VEGFB and VEGFA) may indicate that MAC_MTs participate in blood vessel formation and tissue remodelling (Figure 1G). To gain a wider view of human MAC_MTs, we analyzed an additional dataset5 in which the Macrophage-11 sub-cluster represents MAC_MTs (Figure 2A,B). Metallothionein gene expression was specific to this sub-cluster and present in all 10 vital organs (stomach, kidney, pancreas, colon, lung, ascites, tonsil, liver, breast, and skin) but absent from spleen, blood, and ascites fluid. MAC_MTs were a small percentage of mono-macs, less than 5% in all organs tested (Figure 2C). Metallothionein genes were highly expressed and specific to the Macrophage-11 cluster, as indicated by the high score of our MAC_MT myeloid signature (Figure 2D and Figure S7). These findings indicate that MAC_MTs are a distinctive subset present in multiple vital organs. They were previously reported to be limited to prostate, but have also been localized in lung.6, 7 We used an advanced immunohistochemistry technique,8 consecutive immunohistochemically staining on a single section, to localize MAC_MTs in human liver tissue (Figure 2E). The liver tissues of three patients with advanced liver disease were consecutively stained with an anti-CD68 antibody and an antibody that recognizes MT1 and MT2. Rare CD68+/MT+ cells were identified in all patients and were localized to the regions of ductular reactions that surround regenerative nodules in the cirrhotic liver and support neovascular development. These findings combine with the receptor-ligand data to suggest a role of MAC_MTs in neoangiogenesis. In conclusion, using publicly available scRNA-sequencing data and immunohistochemistry, we transcriptionally profiled MAC_MTs, delineated shared marker genes of MAC_MTs, established their presence in transcriptomic data from multiple vital organs, identified predictors of MAC_MT function, and determined their localization in human liver. Our research suggests that MAC_MTs occur in the majority of vital human organs. Trajectory analysis suggests they differentiate locally within their resident organ, consistent with evidence that corneal tissue upregulates MT genes when cultured with monocyte-derived macrophages.9 Our work sets the stage for mechanistic studies defining the functional role of MAC_MTs in tissue homeostasis and remodelling. Joseph A. Daccache: Conceptualization; investigation; visualization; interpretation and writing. Francis Eng: Conceptualization; investigation. Lei Cao: Methodology; validation. Ning Ma, Stephen C. Ward, Thomas Schiano, Mark Miller and Daniel Herron: Interpretation; resources. Anthony V. Azzara, Steven S. Pullen and Paolo Guarnieri: Conceptualization; Costica Aloman: Conceptualization, interpretation and funding. Andrea D. Branch: Conceptualization; interpretation; writing and funding acquisition. We thank the Mount Sinai pathology core for their contributions to the immunohistochemistry staining. Stephen C. Ward receives grant salary support from Boehringer Ingelheim, Ltd. Daniel Herron has received an honorarium from Intuitive in the past. Anthony V. Azzara and Steven S. Pullen are employees at Boehringer Ingelheim Pharmaceutical Inc. Paolo Guarnieri has stock ownership in 23 & Me. Andrea D. Branch advises the Center for Disease Analysis Foundation and the Icahn School of Medicine at Mount Sinai receives funding from Gilead and Pfizer to support laboratory research. Joseph A. Daccache, Francis Eng, Lei Cao, Ning Ma, Thomas Schiano, Mark Miller and Costica Aloman declare no conflict of interest. This work was funded by Boehringer Ingelheim, NIOSH grants U010H012622 and U010H012263, NIAAA grants R01AA024762 and NCI grant 1U0CA288425, and the Prevent Cancer Foundation. This study utilizes published datasets that have been deidentified and uploaded to the GEO and Zenodo archives. Human samples for IHC staining were collected under GCO numbers 15−1671 and 18−1512. All data was taken from published data sources from GEO `GSE136103, Zenodo https://doi.org/10.5281/zenodo.4059315, and GEO GSE136831. Validation data was taken from the online portal provided by the authors. The code used to generate figures has been uploaded to the GitHub repository daccachejoe/mt-macs. Further requests for code can be requested from the corresponding author Andrea D. Branch upon reasonable request. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.

Viral hepatitis leads to immune-mediated liver injury. The rate of disease progression varies between individuals. We aimed to phenotype immune cells associated with preservation of normal liver function during hepatitis C virus (HCV) infection. Clinical data and specimens were obtained from 19 HCV-infected patients undergoing liver transplantation. Liver and peripheral blood mononuclear cells were isolated and eight subsets of innate immune cells were delineated by multiparameter flow cytometry. Cytokine assays and microarrays were performed. Intrahepatic CD56Bright/CD16- natural killer (NK) cells comprised the only subset correlating with better liver function, i.e., lower bilirubin (p = 0.0002) and lower model for end stage of liver disease scores (p = 0.03). The signature of liver NK cells from HCV-infected patients included genes expressed by NK cells in normal liver and by decidual NK cells. Portal vein blood had a higher concentration of interleukin (IL)-10 than peripheral blood (p = 0.03). LMCs were less responsive to toll-like receptor (TLR) stimulation than PBMCs, with fewer pro-inflammatory gene-expression pathways up-regulated after in vitro exposure to lipopolysaccharide and a TLR-7/8 agonist. Hepatic CD56Bright/CD16- NK cells may be critical for maintaining liver homeostasis. Portal vein IL-10 may prime inhibitory pathways, attenuating TLR signaling and reducing responsiveness to pro-inflammatory stimuli.

Plasmacytoid dendritic cells (pDCs) are “natural” interferon α (IFNα)-producing cells. Despite their importance to antiviral defense, autoimmunity, and ischemic liver graft injury, because DC subsets are rare and heterogeneous, basic questions about liver pDC function and capacity to make cytokines remain unanswered. Previous investigations failed to consistently detect IFNα mRNA in HCV-infected livers, suggesting that pDCs may be incapable of producing IFNα. We used a combination of molecular, biochemical, cytometric, and high-dimensional techniques to analyze DC frequencies/functions in liver and peripheral blood mononuclear cells (PBMCs) of hepatitis C virus (HCV)-infected patients, to examine correlations between DC function and gene expression of matched whole liver tissue and liver mononuclear cells (LMCs), and to determine if pDCs can produce multiple cytokines. T cells often produce multiple cytokines/chemokines but until recently technical limitations have precluded tests of polyfunctionality in individual pDCs. Mass cytometry (CyTOF) revealed that liver pDCs are the only LMC that produces detectable amounts of IFNα in response TLR-7/8 stimulation. Liver pDCs secreted large quantities of IFNα (~2 million molecules of IFNα/cell/hour) and produced more IFNα than PBMCs after stimulation, p = 0.0001. LMCs secreted >14-fold more IFNα than IFNλ in 4 hours. Liver pDC frequency positively correlated with whole liver expression of “IFNα-response” pathway (R2 = 0.58, p = 0.007) and “monocyte surface” signature (R2 = 0.54, p = 0.01). Mass cytometry revealed that IFNα-producing pDCs were highly polyfunctional; >90% also made 2–4 additional cytokines/chemokines of our test set of 10. Liver BDCA1 DCs, but not BDCA3 DCs, were similarly polyfunctional. pDCs from a healthy liver were also polyfunctional. Our data show that liver pDCs retain the ability to make abundant IFNα during chronic HCV infection and produce many other immune modulators. Polyfunctional liver pDCs are likely to be key drivers of inflammation and immune activation during chronic HCV infection.

Hepatitis C virus (HCV) is one of the most prevalent causes of chronic blood‐borne infections worldwide. Despite developments of highly effective treatments, most infected individuals are unaware of their infection. Approximately 75% of infections are in low‐ and middle‐income countries; therefore, continuing research in HCV molecular virology and the development of vaccines and affordable diagnostics is required to reduce the global burden. Various intracellular forms of the HCV nucleocapsid (core) protein are produced in cell culture; these comprise the conventional p21 core and the newly discovered shorter isoforms (minicores). Minicores lack the N‐terminus of p21 core. This study was conducted to determine if minicores are secreted in cell culture and more importantly if they circulate in the blood of individuals infected with HCV. We also developed a new monoclonal antibody that detects minicores targeting a C‐terminal region common to p21 core and minicores. Direct evidence of minicores requires western blot analysis to distinguish the detection of p21 core from minicores. However, the sensitivity for western blot detection of HCV proteins from blood is nil without their prior purification/enrichment from blood. Therefore, we developed a purification method based on a heparin/Mn+2 precipitation of apolipoprotein B‐containing lipoproteins because HCV is thought to circulate as a hybrid lipoviral particle. Minicores are secreted in culture when cells are grown in the presence of human serum. The heparin/Mn+2 precipitate from HCV‐infected cell culture supernatants and from the blood of 4 patients with high‐titer genotype‐1 HCV contained minicores. Conclusion: Minicores are major newly discovered HCV proteins that are secreted and circulate in blood during natural infections. Minicore proteins have translational potential as targets in diagnostic assays and in vaccine development. (Hepatology Communications 2018;2:21–28)

Hepatitis C virus (HCV) is unique among RNA viruses in its ability to establish chronic infection in the majority of exposed adults. HCV persists in the liver despite interferon (IFN)‐stimulated gene (ISG) induction; robust induction actually predicts treatment failure and viral persistence. It is unclear which forms of HCV RNA are associated with ISG induction and IFN resistance during natural infections. To thoroughly delineate HCV RNA populations, we developed conditions that fully separate the strands of long double‐stranded RNA (dsRNA) and allow the released RNAs to be quantified in reverse transcription/polymerase chain reaction assays. These methods revealed that dsRNA, a pathogen‐associated molecular pattern (PAMP), comprised 52% (standard deviation, 28%) of the HCV RNA in the livers of patients with chronic infection. HCV dsRNA was proportionally higher in patients with the unfavorable IL28B TT (rs12979860) genotype. Higher ratios of HCV double‐stranded to single‐stranded RNA (ssRNA) correlated positively with ISG induction. In Huh‐7.5 cells, IFN treatment increased the total amount of HCV dsRNA through a process that required de novo viral RNA synthesis and shifted the ratio of viral dsRNA/ssRNA in favor of dsRNA. This shift was blocked by ribavirin (RBV), an antiviral drug that reduces relapse in HCV patients. Northern blotting established that HCV dsRNA contained genome‐length minus strands. Conclusion : HCV dsRNA is the predominant form in the HCV‐infected liver and has features of both a PAMP and a genomic reservoir. Interferon treatment increased rather than decreased HCV dsRNA. This unexpected finding suggests that HCV produces dsRNA in response to IFN, potentially to antagonize antiviral defenses. (H epatology 2017;66:357–370).

Mutations at positions 70 and/or 91 in the core protein of genotype-1b, hepatitis C virus (HCV) are associated with hepatocellular carcinoma (HCC) risk in Asian patients. To evaluate this in a US population, the relationship between the percentage of 70 and/or 91 mutant HCV quasispecies in baseline serum samples of chronic HCV patients from the HALT-C trial and the incidence of HCC was determined by deep sequencing. Quasispecies percentage cut-points, ≥42% of non-arginine at 70 (non-R70) or ≥98.5% of non-leucine at 91 (non-L91) had optimal sensitivity at discerning higher or lower HCC risk. In baseline samples, 88.5% of chronic HCV patients who later developed HCC and 68.8% of matched HCC-free control patients had ≥42% non-R70 quasispecies (P = 0.06). Furthermore, 30.8% of patients who developed HCC and 54.7% of matched HCC-free patients had quasispecies with ≥98.5% non-L91 (P = 0.06). By Kaplan-Meier analysis, HCC incidence was higher, but not statistically significant, among patients with quasispecies ≥42% non-R70 (P = 0.08), while HCC incidence was significantly reduced among patients with quasispecies ≥98.5% non-L91 (P = 0.01). In a Cox regression model, non-R70 ≥42% was associated with increased HCC risk. This study of US patients indicates the potential utility of HCV quasispecies analysis as a non-invasive biomarker of HCC risk.

Background & Aims: Although patients infected by genotype 1b hepatitis C virus (HCV) with Q(70) and/or M-91 core gene mutations have an almost five-fold increased risk of developing hepatocellular carcinoma (HCC) and increased insulin resistance, the absence of a suitable experimental system has precluded direct experimentation on the effects of these mutations on cellular gene expression.Methods: HuH7 cells were treated long-term with human serum to induce differentiation and to produce a model system for testing high-risk and control HCV. For clinical validation, profiles of infected cells were compared to each other and to those of liver biopsies of patients with early-stage HCV-related cirrhosis followed prospectively for up to 23 years (n = 216).Results: Long-term culture in human serum produced growth-arrested, hepatocyte-like cells whose gene profile overlapped significantly with that of primary human hepatocytes. High-risk (Q(70)/M-91) and control (R-70/L-91) viruses had dramatically different effects on gene expression of these cells. The high-risk virus enhanced expression of pathways associated with cancer and type II diabetes, while the control virus enhanced pathways associated with oxidative phosphorylation. Of special clinical relevance, the transcriptome of cells replicating the high-risk virus correlated significantly with an HCC high-risk profile in patients (Bonferroni-corrected rho = 0.03), whereas no such association was observed for non-HCC-related clinical outcomes.Conclusions: The cell-based system allowed direct head-to-head comparison of HCV variants, and provided experimental support for previous clinical data indicating an oncogenic effect of core gene mutations. This simple experimental system distinguished HCV variants and will enable future mechanistic analysis and exploration of interventional approaches. (C) 2015 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.

327Ashowed enhanced mitochondrial translocation of JNK accompanied by an increase in the release of mitochondrial enzymes, such as apoptosis-inducing factor and endonuclease G, into the cytosol, which is indicative of increased mitochondrial dysfunction and subsequent nuclear DNA fragmentation.Finally, in vitro experiments showed that Gab1-deficient hepatocytes were more susceptible to APAP-induced mitochondrial dysfunction and cell death, suggesting that hepatocyte Gab1 is a direct target of APAP-induced hepatotoxicity.Conclusion: Our current data demonstrate that hepatocyte Gab1 plays a critical role in controlling the balance between hepatocyte death and compensatory hepatocyte proliferation during APAP-induced liver injury.Thus, hepatocyte Gab1 would be a potential therapeutic target for APAP-induced liver injury.