Plasmacytoid dendritic cells: Welcome back to the DC fold.

The presumed common origin of plasmacytoid and conventional dendritic cells has been the contentious subject of recent debate. In this issue of Immunity, Feng et al. employed an inducible cell barcoding system to track clonal relationships and uncovered a surprising close developmental relationship between cDC1s and pDCs.

Early IFN-α signatures and persistent dysfunction are distinguishing features of NK cells in severe COVID-19.

Longitudinal analyses of the innate immune system, including the earliest time points, are essential to understand the immunopathogenesis and clinical course of coronavirus disease (COVID-19). Here, we performed a detailed characterization of natural killer (NK) cells in 205 patients (403 samples; days 2 to 41 after symptom onset) from four independent cohorts using single-cell transcriptomics and proteomics together with functional studies. We found elevated interferon (IFN)-α plasma levels in early severe COVD-19 alongside increased NK cell expression of IFN-stimulated genes (ISGs) and genes involved in IFN-α signaling, while upregulation of tumor necrosis factor (TNF)-induced genes was observed in moderate diseases. NK cells exert anti-SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) activity but are functionally impaired in severe COVID-19. Further, NK cell dysfunction may be relevant for the development of fibrotic lung disease in severe COVID-19, as NK cells exhibited impaired anti-fibrotic activity. Our study indicates preferential IFN-α and TNF responses in severe and moderate COVID-19, respectively, and associates a prolonged IFN-α-induced NK cell response with poorer disease outcome.

Tissue CD14+CD8+ T cells reprogrammed by myeloid cells and modulated by LPS.

The liver is bathed in bacterial products, including lipopolysaccharide transported from the intestinal portal vasculature, but maintains a state of tolerance that is exploited by persistent pathogens and tumours1-4. The cellular basis mediating this tolerance, yet allowing a switch to immunity or immunopathology, needs to be better understood for successful immunotherapy of liver diseases. Here we show that a variable proportion of CD8+ T cells compartmentalized in the human liver co-stain for CD14 and other prototypic myeloid membrane proteins and are enriched in close proximity to CD14high myeloid cells in hepatic zone 2. CD14+CD8+ T cells preferentially accumulate within the donor pool in liver allografts, among hepatic virus-specific and tumour-infiltrating responses, and in cirrhotic ascites. CD14+CD8+ T cells exhibit increased turnover, activation and constitutive immunomodulatory features with high homeostatic IL-10 and IL-2 production ex vivo, and enhanced antiviral/anti-tumour effector function after TCR engagement. This CD14+CD8+ T cell profile can be recapitulated by the acquisition of membrane proteins-including the lipopolysaccharide receptor complex-from mononuclear phagocytes, resulting in augmented tumour killing by TCR-redirected T cells in vitro. CD14+CD8+ T cells express integrins and chemokine receptors that favour interactions with the local stroma, which can promote their induction through CXCL12. Lipopolysaccharide can also increase the frequency of CD14+CD8+ T cells in vitro and in vivo, and skew their function towards the production of chemotactic and regenerative cytokines. Thus, bacterial products in the gut-liver axis and tissue stromal factors can tune liver immunity by driving myeloid instruction of CD8+ T cells with immunomodulatory ability.

Local and systemic responses to SARS-CoV-2 infection in children and adults.

It is not fully understood why COVID-19 is typically milder in children1-3. Here, to examine the differences between children and adults in their response to SARS-CoV-2 infection, we analysed paediatric and adult patients with COVID-19 as well as healthy control individuals (total n = 93) using single-cell multi-omic profiling of matched nasal, tracheal, bronchial and blood samples. In the airways of healthy paediatric individuals, we observed cells that were already in an interferon-activated state, which after SARS-CoV-2 infection was further induced especially in airway immune cells. We postulate that higher paediatric innate interferon responses restrict viral replication and disease progression. The systemic response in children was characterized by increases in naive lymphocytes and a depletion of natural killer cells, whereas, in adults, cytotoxic T cells and interferon-stimulated subpopulations were significantly increased. We provide evidence that dendritic cells initiate interferon signalling in early infection, and identify epithelial cell states associated with COVID-19 and age. Our matching nasal and blood data show a strong interferon response in the airways with the induction of systemic interferon-stimulated populations, which were substantially reduced in paediatric patients. Together, we provide several mechanisms that explain the milder clinical syndrome observed in children.

Blood and immune development in human fetal bone marrow and Down syndrome.

Haematopoiesis in the bone marrow (BM) maintains blood and immune cell production throughout postnatal life. Haematopoiesis first emerges in human BM at 11-12 weeks after conception1,2, yet almost nothing is known about how fetal BM (FBM) evolves to meet the highly specialized needs of the fetus and newborn. Here we detail the development of FBM, including stroma, using multi-omic assessment of mRNA and multiplexed protein epitope expression. We find that the full blood and immune cell repertoire is established in FBM in a short time window of 6-7 weeks early in the second trimester. FBM promotes rapid and extensive diversification of myeloid cells, with granulocytes, eosinophils and dendritic cell subsets emerging for the first time. The substantial expansion of B lymphocytes in FBM contrasts with fetal liver at the same gestational age. Haematopoietic progenitors from fetal liver, FBM and cord blood exhibit transcriptional and functional differences that contribute to tissue-specific identity and cellular diversification. Endothelial cell types form distinct vascular structures that we show are regionally compartmentalized within FBM. Finally, we reveal selective disruption of B lymphocyte, erythroid and myeloid development owing to a cell-intrinsic differentiation bias as well as extrinsic regulation through an altered microenvironment in Down syndrome (trisomy 21).

Rheumatic complications of checkpoint inhibitors: Lessons from autoimmunity.

Immune checkpoint inhibitors are now an established treatment in the management of a range of cancers. Their success means that their use is likely to increase in future in terms of the numbers of patients treated, the indications and the range of immune checkpoints targeted. They function by counteracting immune evasion by the tumor but, as a consequence, can breach self-tolerance at other sites leading to a range of immune-related adverse events. Included among these complications are a range of rheumatologic complications, including inflammatory arthritis and keratoconjunctivitis sicca. These superficially resemble immune-mediated rheumatic diseases (IMRDs) such as rheumatoid arthritis and Sjogren's disease but preliminary studies suggest they are clinically and immunologically distinct entities. However, there appear to be common processes that predispose to the development of both that may inform preventative interventions and predictive tools. Both groups of conditions highlight the centrality of immune checkpoints in controlling tolerance and how it can be restored. Here we will discuss some of these commonalities and differences between rheumatic irAEs and IMRDs.

Decoding human fetal liver haematopoiesis.

Definitive haematopoiesis in the fetal liver supports self-renewal and differentiation of haematopoietic stem cells and multipotent progenitors (HSC/MPPs) but remains poorly defined in humans. Here, using single-cell transcriptome profiling of approximately 140,000 liver and 74,000 skin, kidney and yolk sac cells, we identify the repertoire of human blood and immune cells during development. We infer differentiation trajectories from HSC/MPPs and evaluate the influence of the tissue microenvironment on blood and immune cell development. We reveal physiological erythropoiesis in fetal skin and the presence of mast cells, natural killer and innate lymphoid cell precursors in the yolk sac. We demonstrate a shift in the haemopoietic composition of fetal liver during gestation away from being predominantly erythroid, accompanied by a parallel change in differentiation potential of HSC/MPPs, which we functionally validate. Our integrated map of fetal liver haematopoiesis provides a blueprint for the study of paediatric blood and immune disorders, and a reference for harnessing the therapeutic potential of HSC/MPPs.

Vascular disease persistence in giant cell arteritis: are stromal cells neglected?

Giant cell arteritis (GCA), the most common systemic vasculitis, is characterised by aberrant interactions between infiltrating and resident cells of the vessel wall. Ageing and breach of tolerance are prerequisites for GCA development, resulting in dendritic and T-cell dysfunction. Inflammatory cytokines polarise T-cells, activate resident macrophages and synergistically enhance vascular inflammation, providing a loop of autoreactivity. These events originate in the adventitia, commonly regarded as the biological epicentre of the vessel wall, with additional recruitment of cells that infiltrate and migrate towards the intima. Thus, GCA-vessels exhibit infiltrates across the vascular layers, with various cytokines and growth factors amplifying the pathogenic process. These events activate ineffective repair mechanisms, where dysfunctional vascular smooth muscle cells and fibroblasts phenotypically shift along their lineage and colonise the intima. While high-dose glucocorticoids broadly suppress these inflammatory events, they cause well known deleterious effects. Despite the emerging targeted therapeutics, disease relapse remains common, affecting >50% of patients. This may reflect a discrepancy between systemic and local mediators of inflammation. Indeed, temporal arteries and aortas of GCA-patients can show immune-mediated abnormalities, despite the treatment induced clinical remission. The mechanisms of persistence of vascular disease in GCA remain elusive. Studies in other chronic inflammatory diseases point to the fibroblasts (and their lineage cells including myofibroblasts) as possible orchestrators or even effectors of disease chronicity through interactions with immune cells. Here, we critically review the contribution of immune and stromal cells to GCA pathogenesis and analyse the molecular mechanisms by which these would underpin the persistence of vascular disease.

SARS-CoV-2 infection is associated with anti-desmoglein 2 autoantibody detection.

Post-acute cardiac sequelae, following SARS-CoV-2 infection, are well recognized as complications of COVID-19. We have previously shown the persistence of autoantibodies against antigens in skin, muscle, and heart in individuals following severe COVID-19; the most common staining on skin tissue displayed an inter-cellular cement pattern consistent with antibodies against desmosomal proteins. Desmosomes play a critical role in maintaining the structural integrity of tissues. For this reason, we analyzed desmosomal protein levels and the presence of anti-desmoglein (DSG) 1, 2, and 3 antibodies in acute and convalescent sera from patients with COVID-19 of differing clinical severity. We find increased levels of DSG2 protein in sera from acute COVID-19 patients. Furthermore, we find that DSG2 autoantibody levels are increased significantly in convalescent sera following severe COVID-19 but not in hospitalized patients recovering from influenza infection or healthy controls. Levels of autoantibody in sera from patients with severe COVID-19 were comparable to levels in patients with non-COVID-19-associated cardiac disease, potentially identifying DSG2 autoantibodies as a novel biomarker for cardiac damage. To determine if there was any association between severe COVID-19 and DSG2, we stained post-mortem cardiac tissue from patients who died from COVID-19 infection. This confirmed DSG2 protein within the intercalated discs and disruption of the intercalated disc between cardiomyocytes in patients who died from COVID-19. Our results reveal the potential for DSG2 protein and autoimmunity to DSG2 to contribute to unexpected pathologies associated with COVID-19 infection.

The human liver microenvironment shapes the homing and function of CD4+ T-cell populations.

OBJECTIVE: Tissue-resident memory T cells (TRM) are vital immune sentinels that provide protective immunity. While hepatic CD8+ TRM have been well described, little is known about the location, phenotype and function of CD4+ TRM. DESIGN: We used multiparametric flow cytometry, histological assessment and novel human tissue coculture systems to interrogate the ex vivo phenotype, function and generation of the intrahepatic CD4+ T-cell compartment. We also used leukocytes isolated from human leukocyte antigen (HLA)-disparate liver allografts to assess long-term retention. RESULTS: Hepatic CD4+ T cells were delineated into three distinct populations based on CD69 expression: CD69-, CD69INT and CD69HI. CD69HICD4+ cells were identified as tissue-resident CD4+ T cells on the basis of their exclusion from the circulation, phenotypical profile (CXCR6+CD49a+S1PR1-PD-1+) and long-term persistence within the pool of donor-derived leukcoocytes in HLA-disparate liver allografts. CD69HICD4+ T cells produced robust type 1 polyfunctional cytokine responses on stimulation. Conversely, CD69INTCD4+ T cells represented a more heterogenous population containing cells with a more activated phenotype, a distinct chemokine receptor profile (CX3CR1+CXCR3+CXCR1+) and a bias towards interleukin-4 production. While CD69INTCD4+ T cells could be found in the circulation and lymph nodes, these cells also formed part of the long-term resident pool, persisting in HLA-mismatched allografts. Notably, frequencies of CD69INTCD4+ T cells correlated with necroinflammatory scores in chronic hepatitis B infection. Finally, we demonstrated that interaction with hepatic epithelia was sufficient to generate CD69INTCD4+ T cells, while additional signals from the liver microenvironment were required to generate liver-resident CD69HICD4+ T cells. CONCLUSIONS: High and intermediate CD69 expressions mark human hepatic CD4+ TRM and a novel functionally distinct recirculating population, respectively, both shaped by the liver microenvironment to achieve diverse immunosurveillance.

Establishing the prevalence of common tissue-specific autoantibodies following severe acute respiratory syndrome coronavirus 2 infection.

Coronavirus 19 (COVID-19) has been associated with both transient and persistent systemic symptoms that do not appear to be a direct consequence of viral infection. The generation of autoantibodies has been proposed as a mechanism to explain these symptoms. To understand the prevalence of autoantibodies associated with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, we investigated the frequency and specificity of clinically relevant autoantibodies in 84 individuals previously infected with SARS-CoV-2, suffering from COVID-19 of varying severity in both the acute and convalescent setting. These were compared with results from 32 individuals who were on the intensive therapy unit (ITU) for non-COVID reasons. We demonstrate a higher frequency of autoantibodies in the COVID-19 ITU group compared with non-COVID-19 ITU disease control patients and that autoantibodies were also found in the serum 3-5 months post-COVID-19 infection. Non-COVID patients displayed a diverse pattern of autoantibodies; in contrast, the COVID-19 groups had a more restricted panel of autoantibodies including skin, skeletal muscle and cardiac antibodies. Our results demonstrate that respiratory viral infection with SARS-CoV-2 is associated with the detection of a limited profile of tissue-specific autoantibodies, detectable using routine clinical immunology assays. Further studies are required to determine whether these autoantibodies are specific to SARS-CoV-2 or a phenomenon arising from severe viral infections and to determine the clinical significance of these autoantibodies.

Genomic Characterization of Cholangiocarcinoma in Primary Sclerosing Cholangitis Reveals Therapeutic Opportunities.

BACKGROUND AND AIMS: Lifetime risk of biliary tract cancer (BTC) in primary sclerosing cholangitis (PSC) may exceed 20%, and BTC is currently the leading cause of death in patients with PSC. To open new avenues for management, we aimed to delineate clinically relevant genomic and pathological features of a large panel of PSC-associated BTC (PSC-BTC). APPROACH AND RESULTS: We analyzed formalin-fixed, paraffin-embedded tumor tissue from 186 patients with PSC-BTC from 11 centers in eight countries with all anatomical locations included. We performed tumor DNA sequencing at 42 clinically relevant genetic loci to detect mutations, translocations, and copy number variations, along with histomorphological and immunohistochemical characterization. Regardless of the anatomical localization, PSC-BTC exhibited a uniform molecular and histological characteristic similar to extrahepatic cholangiocarcinoma. We detected a high frequency of genomic alterations typical of extrahepatic cholangiocarcinoma, such as TP53 (35.5%), KRAS (28.0%), CDKN2A (14.5%), and SMAD4 (11.3%), as well as potentially druggable mutations (e.g., HER2/ERBB2). We found a high frequency of nontypical/nonductal histomorphological subtypes (55.2%) and of the usually rare BTC precursor lesion, intraductal papillary neoplasia (18.3%). CONCLUSIONS: Genomic alterations in PSC-BTC include a significant number of putative actionable therapeutic targets. Notably, PSC-BTC shows a distinct extrahepatic morpho-molecular phenotype, independent of the anatomical location of the tumor. These findings advance our understanding of PSC-associated cholangiocarcinogenesis and provide strong incentives for clinical trials to test genome-based personalized treatment strategies in PSC-BTC.

Regulation of S1PR2 by the EBV oncogene LMP1 in aggressive ABC-subtype diffuse large B-cell lymphoma.

The Epstein-Barr virus (EBV) is found almost exclusively in the activated B-cell (ABC) subtype of diffuse large B-cell lymphoma (DLBCL), yet its contribution to this tumour remains poorly understood. We have focused on the EBV-encoded latent membrane protein-1 (LMP1), a constitutively activated CD40 homologue expressed in almost all EBV-positive DLBCLs and which can disrupt germinal centre (GC) formation and drive lymphomagenesis in mice. Comparison of the transcriptional changes that follow LMP1 expression with those that follow transient CD40 signalling in human GC B cells enabled us to define pathogenic targets of LMP1 aberrantly expressed in ABC-DLBCL. These included the down-regulation of S1PR2, a sphingosine-1-phosphate (S1P) receptor that is transcriptionally down-regulated in ABC-DLBCL, and when genetically ablated leads to DLBCL in mice. Consistent with this, we found that LMP1-expressing primary ABC-DLBCLs were significantly more likely to lack S1PR2 expression than were LMP1-negative tumours. Furthermore, we showed that the down-regulation of S1PR2 by LMP1 drives a signalling loop leading to constitutive activation of the phosphatidylinositol-3-kinase (PI3-K) pathway. Finally, core LMP1-PI3-K targets were enriched for lymphoma-related transcription factors and genes associated with shorter overall survival in patients with ABC-DLBCL. Our data identify a novel function for LMP1 in aggressive DLBCL. Copyright © 2019 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Manipulation of Lipid Metabolism During Normothermic Machine Perfusion: Effect of Defatting Therapies on Donor Liver Functional Recovery.

Strategies to increase the use of steatotic donor livers are required to tackle the mortality on the transplant waiting list. We aimed to test the efficacy of pharmacological enhancement of the lipid metabolism of human livers during ex situ normothermic machine perfusion to promote defatting and improve the functional recovery of the organs. Because of steatosis, 10 livers were discarded and were allocated either to a defatting group that had the perfusate supplemented with a combination of drugs to enhance lipid metabolism or to a control group that received perfusion fluid with vehicle only. Steatosis was assessed using tissue homogenate and histological analyses. Markers for lipid oxidation and solubilization, oxidative injury, inflammation, and biliary function were evaluated by enzyme-linked immunosorbent assay, immunohistochemistry, and in-gel protein detection. Treatment reduced tissue triglycerides by 38% and macrovesicular steatosis by 40% over 6 hours. This effect was driven by increased solubility of the triglycerides (P = 0.04), and mitochondrial oxidation as assessed by increased ketogenesis (P = 0.008) and adenosine triphosphate synthesis (P = 0.01) were associated with increased levels of the enzymes acyl-coenzyme A oxidase 1, carnitine palmitoyltransferase 1A, and acetyl-coenzyme A synthetase. Concomitantly, defatted livers exhibited enhanced metabolic functional parameters such as urea production (P = 0.03), lower vascular resistance, lower release of alanine aminotransferase (P = 0.049), and higher bile production (P = 0.008) with a higher bile pH (P = 0.03). The treatment down-regulated the expression of markers for oxidative injury as well as activation of immune cells (CD14; CD11b) and reduced the release of inflammatory cytokines in the perfusate (tumor necrosis factor α; interleukin 1ß). In conclusion, pharmacological enhancement of intracellular lipid metabolism during normothermic machine perfusion decreased the lipid content of human livers within 6 hours. It also improved the intracellular metabolic support to the organs, leading to successful functional recovery and decreased expression of markers of reperfusion injury.

Severe Influenza Is Characterized by Prolonged Immune Activation: Results From the SHIVERS Cohort Study.

Background: The immunologic factors underlying severe influenza are poorly understood. To address this, we compared the immune responses of influenza-confirmed hospitalized individuals with severe acute respiratory illness (SARI) to those of nonhospitalized individuals with influenza-like illness (ILI). Methods: Peripheral blood lymphocytes were collected from 27 patients with ILI and 27 with SARI, at time of enrollment and then 2 weeks later. Innate and adaptive cellular immune responses were assessed by flow cytometry, and serum cytokine levels were assessed by a bead-based assay. Results: During the acute phase, SARI was associated with significantly reduced numbers of circulating myeloid dendritic cells, CD192+ monocytes, and influenza virus-specific CD8+ and CD4+ T cells as compared to ILI. By the convalescent phase, however, most SARI cases displayed continued immune activation characterized by increased numbers of CD16+ monocytes and proliferating, and influenza virus-specific, CD8+ T cells as compared to ILI cases. SARI was also associated with reduced amounts of cytokines that regulate T-cell responses (ie, interleukin 4, interleukin 13, interleukin 12, interleukin 10, and tumor necrosis factor ß) and hematopoiesis (interleukin 3 and granulocyte-macrophage colony-stimulating factor) but increased amounts of a proinflammatory cytokine (tumor necrosis factor α), chemotactic cytokines (MDC, MCP-1, GRO, and fractalkine), and growth-promoting cytokines (PDGFBB/AA, VEGF, and EGF) as compared to ILI. Conclusions: Severe influenza cases showed a delay in the peripheral immune activation that likely led prolonged inflammation, compared with mild influenza cases.

Sphingosine-1-Phosphate Prevents Egress of Hematopoietic Stem Cells From Liver to Reduce Fibrosis.

BACKGROUND & AIMS: There is growing interest in the use of bone marrow cells to treat liver fibrosis, however, little is known about their antifibrotic efficacy or the identity of their effector cell(s). Sphingosine-1-phosphate (S1P) mediates egress of immune cells from the lymphoid organs into the lymphatic vessels; we investigated its role in the response of hematopoietic stem cells (HSCs) to liver fibrosis in mice. METHODS: Purified (c-kit+/sca1+/lin-) HSCs were infused repeatedly into mice undergoing fibrotic liver injury. Chronic liver injury was induced in BoyJ mice by injection of carbon tetrachloride (CCl4) or placement on a methionine-choline-deficient diet. Some mice were irradiated and given transplants of bone marrow cells from C57BL6 mice, with or without the S1P antagonist FTY720; we then studied HSC mobilization and localization. Migration of HSC lines was quantified in Transwell assays. Levels of S1P in liver, bone marrow, and lymph fluid were measured using an enzyme-linked immunosorbent assay. Liver tissues were collected and analyzed by immunohistochemical quantitative polymerase chain reaction and sphingosine kinase activity assays. We performed quantitative polymerase chain reaction analyses of the expression of sphingosine kinase 1 and 2, sphingosine-1-phosphate lyase 1, and sphingosine-1-phosphate phosphatase 1 in normal human liver and cirrhotic liver from patients with alcohol-related liver disease (n = 6). RESULTS: Infusions of HSCs into mice with liver injury reduced liver scarring based on picrosirius red staining (49.7% reduction in mice given HSCs vs control mice; P < .001), and hepatic hydroxyproline content (328 mg/g in mice given HSCs vs 428 mg/g in control mice; P < .01). HSC infusion also reduced hepatic expression of α-smooth muscle actin (0.19 ± 0.007-fold compared with controls; P < .0001) and collagen type I α 1 chain (0.29 ± 0.17-fold compared with controls; P < .0001). These antifibrotic effects were maintained with infusion of lymphoid progenitors that lack myeloid potential and were associated with increased numbers of recipient neutrophils and macrophages in liver. In studies of HSC cell lines, we found HSCs to recruit monocytes, and this process to require C-C motif chemokine receptor 2. In fibrotic liver tissue from mice and patients, hepatic S1P levels increased owing to increased hepatic sphingosine kinase-1 expression, which contributed to a reduced liver:lymph S1P gradient and limited HSC egress from the liver. Mice given the S1P antagonist (FTY720) with HSCs had increased hepatic retention of HSCs (1697 ± 247 cells in mice given FTY720 vs 982 ± 110 cells in controls; P < .05), and further reductions in fibrosis. CONCLUSIONS: In studies of mice with chronic liver injury, we showed the antifibrotic effects of repeated infusions of purified HSCs. We found that HSCs promote recruitment of endogenous macrophages and neutrophils. Strategies to reduce SIP signaling and increase retention of HSCs in the liver could increase their antifibrotic activities and be developed for treatment of patients with liver fibrosis.

The Reactive Oxygen Species-Mitophagy Signaling Pathway Regulates Liver Endothelial Cell Survival During Ischemia/Reperfusion Injury.

Ischemia/reperfusion injury (IRI) is the main cause of complications following liver transplantation. Reactive oxygen species (ROS) were thought to be the main regulators of IRI. However, recent studies demonstrate that ROS activate the cytoprotective mechanism of autophagy promoting cell survival. Liver IRI initially damages the liver endothelial cells (LEC), but whether ROS-autophagy promotes cell survival in LEC during IRI is not known. Primary human LEC were isolated from human liver tissue and exposed to an in vitro model of IRI to assess the role of autophagy in LEC. The role of autophagy during liver IRI in vivo was assessed using a murine model of partial liver IRI. During IRI, ROS specifically activate autophagy-related protein (ATG) 7 promoting autophagic flux and the formation of LC3B-positive puncta around mitochondria in primary human LEC. Inhibition of ROS reduces autophagic flux in LEC during IRI inducing necrosis. In addition, small interfering RNA knockdown of ATG7 sensitized LEC to necrosis during IRI. In vivo murine livers in uninjured liver lobes demonstrate autophagy within LEC that is reduced following IRI with concomitant reduction in autophagic flux and increased cell death. In conclusion, these findings demonstrate that during liver IRI ROS-dependent autophagy promotes the survival of LEC, and therapeutic targeting of this signaling pathway may reduce liver IRI following transplantation.

Development of Clinical Criteria for Functional Assessment to Predict Primary Nonfunction of High-Risk Livers Using Normothermic Machine Perfusion.

Increased use of high-risk allografts is critical to meet the demand for liver transplantation. We aimed to identify criteria predicting viability of organs, currently declined for clinical transplantation, using functional assessment during normothermic machine perfusion (NMP). Twelve discarded human livers were subjected to NMP following static cold storage. Livers were perfused with a packed red cell-based fluid at 37°C for 6 hours. Multilevel statistical models for repeated measures were employed to investigate the trend of perfusate blood gas profiles and vascular flow characteristics over time and the effect of lactate-clearing (LC) and non-lactate-clearing (non-LC) ability of the livers. The relationship of lactate clearance capability with bile production and histological and molecular findings were also examined. After 2 hours of perfusion, median lactate concentrations were 3.0 and 14.6 mmol/L in the LC and non-LC groups, respectively. LC livers produced more bile and maintained a stable perfusate pH and vascular flow >150 and 500 mL/minute through the hepatic artery and portal vein, respectively. Histology revealed discrepancies between subjectively discarded livers compared with objective findings. There were minimal morphological changes in the LC group, whereas non-LC livers often showed hepatocellular injury and reduced glycogen deposition. Adenosine triphosphate levels in the LC group increased compared with the non-LC livers. We propose composite viability criteria consisting of lactate clearance, pH maintenance, bile production, vascular flow patterns, and liver macroscopic appearance. These have been tested successfully in clinical transplantation. In conclusion, NMP allows an objective assessment of liver function that may reduce the risk and permit use of currently unused high-risk livers.

Autotaxin-lysophosphatidic acid receptor signalling regulates hepatitis C virus replication.

BACKGROUND & AIMS: Chronic hepatitis C is a global health problem with an estimated 170 million hepatitis C virus (HCV) infected individuals at risk of progressive liver disease and hepatocellular carcinoma (HCC). Autotaxin (ATX, gene name: ENPP2) is a phospholipase with diverse roles in the physiological and pathological processes including inflammation and oncogenesis. Clinical studies have reported increased ATX expression in chronic hepatitis C, however, the pathways regulating ATX and its role in the viral life cycle are not well understood. METHODS: In vitro hepatocyte and ex vivo liver culture systems along with chimeric humanized liver mice and HCC tissue enabled us to assess the interplay between ATX and the HCV life cycle. RESULTS: HCV infection increased hepatocellular ATX RNA and protein expression. HCV infection stabilizes hypoxia inducible factors (HIFs) and we investigated a role for these transcription factors to regulate ATX. In vitro studies show that low oxygen increases hepatocellular ATX expression and transcriptome analysis showed a positive correlation between ATX mRNA levels and hypoxia gene score in HCC tumour tissue associated with HCV and other aetiologies. Importantly, inhibiting ATX-lysophosphatidic acid (LPA) signalling reduced HCV replication, demonstrating a positive role for this phospholipase in the viral life cycle. LPA activates phosphoinositide-3-kinase that stabilizes HIF-1α and inhibiting the HIF signalling pathway abrogates the pro-viral activity of LPA. CONCLUSIONS: Our data support a model where HCV infection increases ATX expression which supports viral replication and HCC progression. LAY SUMMARY: Chronic hepatitis C is a global health problem with infected individuals at risk of developing liver disease that can progress to hepatocellular carcinoma. Autotaxin generates the biologically active lipid lysophosphatidic acid that has been reported to play a tumorigenic role in a wide number of cancers. In this study we show that hepatitis C virus infection increases autotaxin expression via hypoxia inducible transcription factor and provides an environment in the liver that promotes fibrosis and liver injury. Importantly, we show a new role for lysophosphatidic acid in positively regulating hepatitis C virus replication.

High resolution sequencing of hepatitis C virus reveals limited intra-hepatic compartmentalization in end-stage liver disease.

BACKGROUND & AIMS: The high replication and mutation rate of hepatitis C virus (HCV) results in a heterogeneous population of viral sequences in vivo. HCV replicates in the liver and infected hepatocytes occur as foci surrounded by uninfected cells that may promote compartmentalization of viral variants. Given recent reports showing interferon stimulated gene (ISG) expression in chronic hepatitis C, we hypothesized that local interferon responses may limit HCV replication and evolution. METHODS: To investigate the spatial influence of liver architecture on viral replication we measured HCV RNA and ISG mRNA from each of the 8 Couinaud segments of the liver from 21 patients undergoing liver transplant. RESULTS: HCV RNA and ISG mRNA levels were comparable across all sites from an individual liver but showed up to 500-fold difference between patients. Importantly, there was no association between ISG and HCV RNA expression across all sites in the liver or plasma. Deep sequencing of HCV RNA isolated from the 8 hepatic sites from two subjects showed a similar distribution of viral quasispecies across the liver and uniform sequence diversity. Single genome amplification of HCV E1E2-envelope clones from 6 selected patients at 2 hepatic sites supported these data and showed no evidence for HCV compartmentalization. CONCLUSIONS: We found no differences between the hepatic and plasma viral quasispecies in all patients sampled. We conclude that in end-stage liver disease HCV RNA levels and the genetic pool of HCV envelope sequences are indistinguishable between distant sites in the liver and plasma, arguing against viral compartmentalization. LAY SUMMARY: HCV is an RNA virus that exists as a quasispecies of closely related genomes that are under continuous selection by host innate and adaptive immune responses and antiviral drug therapy. The primary site of HCV replication is the liver and yet our understanding of the spatial distribution of viral variants within the liver is limited. High resolution sequencing of HCV and monitoring of innate immune responses at multiple sites across the liver identified a uniform pattern of diversity and argues against viral compartmentalization.