[Current diagnostics and treatment of portal hypertension]. / Aktuelle Diagnostik und Therapie der portalen Hypertension.

In industrial nations portal hypertension is mostly a consequence of liver cirrhosis and is a prerequisite for complications, such as esophageal varices and ascites. The pathophysiology of portal hypertension is complex. It is defined as an increase in the hepatovenous pressure gradient to > 5 mm Hg, with complications to be expected at ≥ 10 mm Hg. Measurement of the pressure of the hepatic vein occlusion is the gold standard for estimating portal pressure but this is not very practical. Liver elastography, in particular, has proven to be an effective noninvasive tool to identify patients with clinically significant portal hypertension (CSPH). Current treatment concepts address the CSPH even before the onset of complications to reduce the likelihood of decompensation. In addition to beta blockers, a transjugular intrahepatic portosystemic shunt is the most important procedure to lower portal vein pressure and enables an improvement in the prognosis of selected patients.

Weakly supervised end-to-end artificial intelligence in gastrointestinal endoscopy.

Artificial intelligence (AI) is widely used to analyze gastrointestinal (GI) endoscopy image data. AI has led to several clinically approved algorithms for polyp detection, but application of AI beyond this specific task is limited by the high cost of manual annotations. Here, we show that a weakly supervised AI can be trained on data from a clinical routine database to learn visual patterns of GI diseases without any manual labeling or annotation. We trained a deep neural network on a dataset of N = 29,506 gastroscopy and N = 18,942 colonoscopy examinations from a large endoscopy unit serving patients in Germany, the Netherlands and Belgium, using only routine diagnosis data for the 42 most common diseases. Despite a high data heterogeneity, the AI system reached a high performance for diagnosis of multiple diseases, including inflammatory, degenerative, infectious and neoplastic diseases. Specifically, a cross-validated area under the receiver operating curve (AUROC) of above 0.70 was reached for 13 diseases, and an AUROC of above 0.80 was reached for two diseases in the primary data set. In an external validation set including six disease categories, the AI system was able to significantly predict the presence of diverticulosis, candidiasis, colon and rectal cancer with AUROCs above 0.76. Reverse engineering the predictions demonstrated that plausible patterns were learned on the level of images and within images and potential confounders were identified. In summary, our study demonstrates the potential of weakly supervised AI to generate high-performing classifiers and identify clinically relevant visual patterns based on non-annotated routine image data in GI endoscopy and potentially other clinical imaging modalities.

Immunomodulatory receptor VSIG4 is released during spontaneous bacterial peritonitis and predicts short-term mortality.

BACKGROUND & AIMS: V-set Ig-domain-containing 4 (VSIG4) is an immunomodulatory macrophage complement receptor modulating innate and adaptive immunity and affecting the resolution of bacterial infections. Given its expression on peritoneal macrophages (PMs), we hypothesised a prognostic role of peritoneal VSIG4 concentrations in patients with spontaneous bacterial peritonitis (SBP). METHODS: We isolated PMs from patients with cirrhosis and analysed VSIG4 expression and release by flow cytometry, quantitative real-time PCR, ELISA, and confocal microscopy. We measured soluble VSIG4 concentrations in ascites from 120 patients with SBP and 40 patients without SBP and investigated the association of soluble VSIG4 in ascites with 90-day survival after SBP using Kaplan-Meier statistics, Cox regression, and competing-risks regression analysis. RESULTS: VSIG4 expression was high on resting, large PMs, which co-expressed CD206, CD163, and tyrosine-protein kinase Mer (MERTK). VSIG4 gene expression in PMs decreased in patients with SBP and normalised after resolution. During SBP, VSIG4hi PMs were depleted (25% vs. 57%; p <0.001) and soluble VSIG4 in ascites were higher in patients with SBP than in patients without (0.73 vs. 0.35 µg/ml; p <0.0001). PM activation by Toll-like receptor (TLR) agonists or infection with live bacteria in vitro resulted in a loss of surface VSIG4 and the release of soluble VSIG4. Mechanistically, shedding of VSIG4 from PMs was protease-dependent and susceptible to microtubule transport inhibition. Soluble VSIG4 in ascites exceeded serum concentrations and correlated with serum creatinine, model for end-stage liver disease score and C-reactive protein during SBP. Concentrations of 1.0206 µg/ml or higher indicated increased 90-day mortality (hazard ratio 1.70; 95% CI 1.01-2.86; p = 0.046). CONCLUSIONS: VSIG4 is released from activated PMs into ascites during SBP. Higher peritoneal VSIG4 levels indicate patients with organ failure and poor prognosis. LAY SUMMARY: Patients with liver cirrhosis who develop ascites have an increased risk of infection and mortality. Our study shows that in patients with infected ascites, the complement receptor VSIG4 is released by resident macrophages into the abdominal fluid where it can be measured. Patients with elevated levels of this protein in ascites are at high risk of dying within 90 days.

Balance between macrophage migration inhibitory factor and sCD74 predicts outcome in patients with acute decompensation of cirrhosis.

BACKGROUND & AIMS: Macrophage migration inhibitory factor (MIF) is an inflammatory cytokine and an important regulator of innate immune responses. We hypothesised that serum concentrations of MIF are associated with disease severity and outcome in patients with decompensated cirrhosis and acute-on-chronic liver failure (ACLF). METHODS: Circulating concentrations of MIF and its soluble receptor CD74 (sCD74) were determined in sera from 292 patients with acute decompensation of cirrhosis defined as new onset or worsening of ascites requiring hospitalisation. Of those, 78 (27%) had ACLF. Short-term mortality was assessed 90 days after inclusion. RESULTS: Although serum concentrations of MIF and sCD74 did not correlate with liver function parameters or ACLF, higher MIF (optimum cut-off >2.3 ng/ml) and lower concentrations of sCD74 (optimum cut-off <66.5 ng/ml) both indicated poorer 90-day transplant-free survival in univariate analyses (unadjusted hazard ratio [HR] 2.01 [1.26-3.22]; p = 0.004 for MIF; HR 0.59 [0.38-0.92]; p = 0.02 for sCD74) and after adjustment in multivariable models. Higher MIF concentrations correlated with surrogates of systemic inflammation (white blood cells, p = 0.005; C-reactive protein, p = 0.05) and were independent of genetic MIF promoter polymorphisms. Assessment of MIF plasma concentrations in portal venous blood and matched blood samples from the right atrium in a second cohort of patients undergoing transjugular intrahepatic portosystemic shunt insertion revealed a transhepatic MIF gradient with higher concentrations in the right atrial blood. CONCLUSIONS: Serum concentrations of MIF and its soluble receptor CD74 predict 90-day transplant-free survival in patients with acute decompensation of cirrhosis. This effect was independent of liver function and genetic predispositions, but rather reflected systemic inflammation. Therefore, MIF and sCD74 represent promising prognostic markers beyond classical scoring systems in patients at risk of ACLF. LAY SUMMARY: Inflammatory processes contribute to the increased risk of death in patients with cirrhosis and ascites. We show that patients with high serum levels of the inflammatory cytokine macrophage migration inhibitory factor (MIF) alongside low levels of its binding receptor sCD74 in blood indicate an increased mortality risk in patients with ascites. The cirrhotic liver is a relevant source of elevated circulating MIF levels.

Intestinal dysbiosis augments liver disease progression via NLRP3 in a murine model of primary sclerosing cholangitis.

OBJECTIVE: There is a striking association between human cholestatic liver disease (CLD) and inflammatory bowel disease. However, the functional implications for intestinal microbiota and inflammasome-mediated innate immune response in CLD remain elusive. Here we investigated the functional role of gut-liver crosstalk for CLD in the murine Mdr2 knockout (Mdr2-/-) model resembling human primary sclerosing cholangitis (PSC). DESIGN: Male Mdr2-/-, Mdr2-/- crossed with hepatocyte-specific deletion of caspase-8 (Mdr2-/- /Casp8∆hepa) and wild-type (WT) control mice were housed for 8 or 52 weeks, respectively, to characterise the impact of Mdr2 deletion on liver and gut including bile acid and microbiota profiling. To block caspase activation, a pan-caspase inhibitor (IDN-7314) was administered. Finally, the functional role of Mdr2-/- -associated intestinal dysbiosis was studied by microbiota transfer experiments. RESULTS: Mdr2-/- mice displayed an unfavourable intestinal microbiota signature and pronounced NLRP3 inflammasome activation within the gut-liver axis. Intestinal dysbiosis in Mdr2-/- mice prompted intestinal barrier dysfunction and increased bacterial translocation amplifying the hepatic NLRP3-mediated innate immune response. Transfer of Mdr2-/- microbiota into healthy WT control mice induced significant liver injury in recipient mice, highlighting the causal role of intestinal dysbiosis for disease progression. Strikingly, IDN-7314 dampened inflammasome activation, ameliorated liver injury, reversed serum bile acid profile and cholestasis-associated microbiota signature. CONCLUSIONS: MDR2-associated cholestasis triggers intestinal dysbiosis. In turn, translocation of endotoxin into the portal vein and subsequent NLRP3 inflammasome activation contribute to higher liver injury. This process does not essentially depend on caspase-8 in hepatocytes, but can be blocked by IDN-7314.

Inactivation of caspase 8 in liver parenchymal cells confers protection against murine obstructive cholestasis.

BACKGROUND & AIMS: Caspase 8 (CASP8) is the apical initiator caspase in death receptor-mediated apoptosis. Strong evidence for a link between death receptor signaling pathways and cholestasis has recently emerged. Herein, we investigated the role of CASP8-dependent and independent pathways during experimental cholestasis. METHODS: Liver injury was characterized in a cohort of human sera (n = 28) and biopsies from patients with stage IV primary biliary cholangitis. In parallel, mice with either specific deletion of Casp8 in liver parenchymal cells (Casp8Δhepa) or hepatocytes (Casp8Δhep), and mice with constitutive Ripk3 (Ripk3-/-) deletion, were subjected to surgical ligation of the common bile duct (BDL) from 2 to 28 days. Floxed (Casp8fl/fl) and Ripk3+/+ mice were used as controls. Moreover, the pan-caspase inhibitor IDN-7314 was used, and cell death mechanisms were studied in primary isolated hepatocytes. RESULTS: Overexpression of activated caspase 3, CASP8 and RIPK3 was characteristic of liver explants from patients with primary biliary cholangitis. Twenty-eight days after BDL, Casp8Δhepamice showed decreased necrotic foci, serum aminotransferase levels and apoptosis along with diminished compensatory proliferation and ductular reaction. These results correlated with a decreased inflammatory profile and ameliorated liver fibrogenesis. A similar phenotype was observed in Ripk3-/- mice. IDN-7314 treatment decreased CASP8 levels but failed to prevent BDL-induced cholestasis, independently of CASP8 in hepatocytes. CONCLUSION: These findings show that intervention against CASP8 in liver parenchymal cells - specifically in cholangiocytes - might be a beneficial option for treating obstructive cholestasis, while broad pan-caspase inhibition might trigger undesirable side effects. LAY SUMMARY: Loss of caspase 8 - a protein involved in programmed cell death - in liver parenchymal cells protects against experimental cholestasis. Therefore, specific pharmacological intervention against caspase 8 might be a valid alternative for the treatment of obstructive cholestasis in the clinic, whereas broad pan-caspase inhibiting drugs might trigger undesirable side effects.

Inhibition of Caspase-8 does not protect from alcohol-induced liver apoptosis but alleviates alcoholic hepatic steatosis in mice.

Hepatic apoptosis is involved in the progression of alcoholic liver disease (ALD). Caspase-8, the apical initiator in death receptor-mediated apoptosis, has been implicated in acute liver injury and in non-alcoholic steatohepatitis. However, the relevance of Caspase-8 in the pathogenesis of ALD remains unclear. In the present study, we investigated the impact of Caspase-8 in human and murine alcohol-induced apoptosis and in ALD. We investigated human samples from ALD patients, primary mouse hepatocytes, and hepatocyte-specific Caspase-8 knockout (Casp8Δhepa) mice in acute and chronic models of ethanol (EtOH) administration. Caspase-8 activation was detected in liver biopsies from ALD patients, as well as in livers of wild-type (WT) mice after chronic ethanol feeding for 8 weeks using the Lieber-DeCarli model. Lack of Caspase-8 expression in Casp8Δhepa animals failed to prevent alcohol-induced liver damage and apoptosis. Instead, inhibition of Caspase-8 shifted the ethanol-induced death signals towards pronounced activation of the intrinsic, mitochondria-dependent apoptosis pathway in Casp8Δhepa livers involving enhanced release of cytochrome c, stronger Caspase-9 activation and specific morphological changes of mitochondria. In vitro and in vivo intervention using a pan-caspase inhibitor markedly attenuated alcohol-induced hepatocyte damage in a Caspase-8-independent manner. Surprisingly, EtOH-fed Casp8Δhepa mice displayed significantly attenuated steatosis and reduced hepatic triglyceride and free fatty acids content. Caspase-8 is dispensable for alcohol-induced apoptosis, but plays an unexpected role for alcohol-dependent fat metabolism. We provide evidence that simultaneous inhibition of extrinsic and intrinsic apoptosis signaling using pan-caspase inhibitors in vivo might be an optimal approach to treat alcohol-induced liver injury.

Impaired Transmigration of Myeloid-Derived Suppressor Cells across Human Sinusoidal Endothelium Is Associated with Decreased Expression of CD13.

Human monocytic myeloid-derived suppressor cells (MO-MDSCs) within the hepatic compartment suppress inflammation and impair immune surveillance in liver cancer. It is currently not known whether recruitment of MO-MDSCs from blood via hepatic sinusoidal endothelium (HSEC) contributes to their enrichment within the hepatic compartment. We compared the transmigratory potential of MO-MDSCs and monocytes after adhesion to hepatic endothelial monolayers in flow-based assays that mimic in vivo shear stress in the sinusoids. Despite comparable binding to HSEC monolayers, proportionally fewer MO-MDSCs underwent transendothelial migration, indicating that the final steps of extravasation, where actin polymerization plays an important role, are impaired in MO-MDSCs. In this article, we found reduced levels of CD13 on MO-MDSCs, which has recently been reported to control cell motility in monocytes, alongside reduced VLA-4 expression, an integrin predominantly involved in adherence to the apical side of the endothelium. CD13 and VLA-4 blocking and activating Abs were used in flow-based adhesion assays, live-cell imaging of motility, and actin polymerization studies to confirm a role for CD13 in impaired MO-MDSC transmigration. These findings indicate that CD13 significantly contributes to tissue infiltration by MO-MDSCs and monocytes, thereby contributing to the pathogenesis of hepatic inflammation.

Low serum transferrin correlates with acute-on-chronic organ failure and indicates short-term mortality in decompensated cirrhosis.

BACKGROUND & AIMS: Iron represents an essential, but potentially harmful micronutrient, whose regulation has been associated with poor outcome in liver disease. Its homeostasis is tightly linked to oxidative stress, bacterial infections and systemic inflammation. To study the prognostic short-term significance of iron parameters in a cohort study of patients with decompensation of cirrhosis at risk of acute-on-chronic liver failure (ACLF). METHODS: Ferritin, transferrin, iron, transferrin saturation (TSAT) and hepcidin were determined in sera from 292 German patients hospitalized for decompensation of cirrhosis with ascites, of which 78 (27%) had ACLF. Short-term mortality was prospectively assessed 30 and 90 days after inclusion. RESULTS: Transferrin concentrations were significantly lower, whereas ferritin and TSAT were higher in patients with ACLF compared to patients without ACLF (P≤.006). Transferrin, TSAT and ferritin differentially correlated with the severity of organ failure, active alcoholism and surrogates of systemic inflammation and macrophage activation. As compared with survivors, 30-day non-survivors displayed lower serum transferrin (P=.0003) and higher TSAT (P=.003), whereas 90-day non-survivors presented with higher ferritin (P=.03) and lower transferrin (P=.02). Lower transferrin (continuous or dichotomized at 87 mg/dL) and consecutively higher TSAT (continuous or dichotomized >41%) indicated increased mortality within 30 days and remained significant after adjustment for organ failure and inflammation in multivariate regression models and across subgroups of patients. CONCLUSION: Among the investigated indicators of iron metabolism, serum transferrin concentration was the best indicator of organ failure and an independent predictor of short-term mortality at 30 days.

Keratin 23 is a stress-inducible marker of mouse and human ductular reaction in liver disease.

BACKGROUND & AIMS: Keratins (K) constitute the epithelial intermediate filaments. Among them, K7/K19 are widely used markers of the regenerative liver response termed ductular reaction (DR) that consists of activated biliary epithelial cells (BECs) and hepatic progenitor cells (HPCs) and correlates with liver disease severity. In the present study we aimed to characterize K23 in the liver. METHODS: We analyzed the expression and localization of K23 in the digestive system under basal conditions as well as in various human and mouse liver diseases/stress models. Cell culture studies were used to study factors regulating K23 expression. RESULTS: In untreated mice, K23 was restricted to biliary epithelia. It was (together with K7/K19) markedly upregulated in three different DR/cholestatic injury models, i.e., multidrug resistance protein 2 (Mdr2) knockouts, animals treated with 3,5-diethoxycarbonyl-1,4-dihydrocollidine or subjected to bile duct ligation. K23 levels correlated with the DR marker Fn14 and immunofluorescence staining showed a distinct co-localization with K7/K19. In chronic human liver disease, K23 expression increased in patients with a more prominent inflammation/fibrosis. A dramatic upregulation (>200times) was observed in patients with acute liver failure (ALF) and end-stage primary biliary cholangitis (PBC). Patients with alcoholic liver cirrhosis displayed increased K23 serum levels. In primary hepatocytes as well as hepatobiliary cell lines, treatment with TNF-related weak inducer of apoptosis (TWEAK), and the type I acute phase inducer interleukin (IL)-1ß but not the type II inducer IL-6 elevated K23 expression. CONCLUSIONS: K23 represents a specific, stress-inducible DR marker, whose levels correlate with liver disease severity. K23 may represent a useful non-invasive DR marker. LAY SUMMARY: Ductular reaction represents a basic response to liver injury and correlates with liver disease severity. Our study identifies K23 as a novel ductular reaction marker in mice and humans.

Enhanced expression of c-myc in hepatocytes promotes initiation and progression of alcoholic liver disease.

BACKGROUND & AIMS: Progression of alcoholic liver disease (ALD) can be influenced by genetic factors, which potentially include specific oncogenes and tumor suppressors. In the present study, we tested the hypothesis that aberrant expression of the proto-oncogene c-myc might exert a crucial role in the development of ALD. METHODS: Expression of c-myc was measured in biopsies of patients with ALD by quantitative real-time PCR and immunohistochemistry. Mice with transgenic expression of c-myc in hepatocytes (alb-myc(tg)) and wild-type (WT) controls were fed either control or ethanol (EtOH) containing Lieber-DeCarli diet for 4weeks to induce ALD. RESULTS: Hepatic c-myc was strongly upregulated in human patients with advanced ALD and in EtOH-fed WT mice. Transcriptome analysis indicated deregulation of pathways involved in ER-stress, p53 signaling, hepatic fibrosis, cell cycle regulation, ribosomal synthesis and glucose homeostasis in EtOH-fed alb-myc(tg) mice. Transgenic expression of c-myc in hepatocytes with simultaneous EtOH-uptake led to early ballooning degeneration, increased liver collagen deposition and hepatic lipotoxicity, together with excessive CYP2E1-derived reactive oxygen species (ROS) production. Moreover, EtOH-fed alb-myc(tg) mice exhibited substantial changes in mitochondrial morphology associated with energy dysfunction. Pathway analysis revealed that elevated c-myc expression and ethanol uptake synergistically lead to strong AKT activation, Mdm2 phosphorylation and as a consequence to inhibition of p53. CONCLUSIONS: Expression of c-myc and EtOH-uptake synergistically accelerate the progression of ALD most likely due to loss of p53-dependent protection. Thus, c-myc is a new potential marker for the early detection of ALD and identification of risk patients.

Bidirectional transendothelial migration of monocytes across hepatic sinusoidal endothelium shapes monocyte differentiation and regulates the balance between immunity and tolerance in liver.

UNLABELLED: Monocytes are versatile cells that can fulfill proinflammatory and anti-inflammatory functions when recruited to the liver. Recruited monocytes differentiate into tissue macrophages and dendritic cells, which sample antigens and migrate to lymph nodes to elicit T-cell responses. The signals that determine monocyte differentiation and the role of hepatic sinusoidal endothelial cells (HSECs) in this process are poorly understood. HSECs are known to modulate T-cell activation, which led us to investigate whether transendothelial migration of monocytes across HSECs influences their phenotype and function. Subsets of blood-derived monocytes were allowed to transmigrate across human HSECs into a collagen matrix. Most migrated cells remained in the subendothelial matrix, but ~10% underwent spontaneous basal to apical transendothelial migration. The maturation, cytokine secretion, and T-cell stimulatory capacity of reverse transmigrating (RT) and subendothelial (SE) monocytes were compared. SE monocytes were mainly CD16(-) , whereas 75%-80% of RT monocytes were CD16(+) . SE monocytes derived from the CD14(++) CD16(-) subset and exhibited high phagocytic activity, whereas RT monocytes originated from CD14(++) CD16(+) and CD14(+) CD16(++) monocytes, displayed an immature dendritic cell-like phenotype (CD11c(pos) HLA-DR(pos) CD80lo CD86lo ), and expressed higher levels of chemokine (C-C motif) receptor 8. Consistent with a dendritic cell phenotype, RT monocytes secreted inflammatory cytokines and induced antigen-specific CD4(+) T-cell activation. In contrast, SE monocytes suppressed T-cell proliferation and activation and exhibited endotoxin tolerance. Transcriptome analysis underscored the functional differences between SE and RT monocytes. CONCLUSIONS: Migration across HSECs shapes the subsequent fate of monocytes, giving rise to anergic macrophage-like cells in tissue and the release of immunocompetent pre-dendritic cells into the circulation.

Chemokine (C-X-C motif) ligand 11 levels predict survival in cirrhotic patients with transjugular intrahepatic portosystemic shunt.

BACKGROUND & AIMS: Chemokines, such as CXCR3-ligands, have been identified to play an important role during hepatic injury, inflammation and fibrosis. While CXCL9 is associated with survival in patients receiving transjugular intrahepatic portosystemic shunt (TIPS), the role of CXCL11 in severe portal hypertension remains unknown. METHODS: CXCL11-levels were measured in 136 patients with liver diseases, and 63 healthy controls. In further 47 cirrhotic patients receiving TIPS, CXCL11 levels were measured in portal and hepatic veins at TIPS insertion by cytometric bead array. CXCL11-levels were measured in 23 patients in cubital vein and right atrium, whereas in 24 patients in portal and hepatic blood at an invasive reevaluation. RESULTS: CXCL11-levels were increased with the severity of liver fibrosis. CXCL11-levels from portal, hepatic and cubital veins and right atrium showed a highly significant correlation among each other in these patients. Furthermore, levels of CXCL11 from the right atrium were significantly higher than those from cubital vein. Interestingly, patients with alcoholic cirrhosis had significantly lower CXCL11-levels, than other aetiologies of cirrhosis. After TIPS, CXCL11 levels correlated with the degree of portal pressure and patients with higher CXCL11-levels in portal and hepatic veins showed higher mortality. Multivariate analysis revealed hepatic CXCL11-levels before TIPS, creatinine and age as independent predictors for survival in TIPS patients, whereas MELD score and low portal CXCL11-levels after TIPS predicted long-term survival. CONCLUSION: CXCL11 levels are mainly increased in patients with non-alcoholic cirrhosis and high portal pressure. Moreover, levels of CXCL11 might predict long-time survival of cirrhotic patients bearing TIPS.

In search of the magic bullet: can liver inflammation and fibrosis be reversed with medications?

Recent clinical studies comprising patients successfully treated for viral hepatitis have shown that liver fibrogenesis may be reverted, even at later stages including during bridging fibrosis and cirrhosis. Intensive research has identified numerous potential novel targets in liver disease. Multiple innovative compounds have now entered clinical trials, mostly in non-alcoholic steatohepatitis (NASH) and NASH-associated cirrhosis due to their outstanding epidemiological relevance. In general, regression from liver fibrosis follows four major mechanistic principles: termination of chronic damage, shifting the cellular bias from inflammation to resolution, deactivation of myofibroblasts and direct matrix degradation. Obeying these principles, several promising approaches are currently evaluated, for example, targeting inflammatory macrophages via inhibition of chemokine CCL2, its receptor CCR2 or galectin-3, bone marrow-derived cell transfer, or antibodies against matrix-stabilizing lysyl oxidase-like-2. The ongoing trials will reveal which of the many potential targets prove to have clinical efficacy, bearing in mind that fibrosis reversibility is less likely to be achieved in humans than in animal models.

CMV infection of human sinusoidal endothelium regulates hepatic T cell recruitment and activation.

BACKGROUND & AIMS: Human cytomegalovirus infection (HCMV) is associated with an increased morbidity after liver transplantation, by facilitating allograft rejection and accelerating underlying hepatic inflammation. We hypothesized that human hepatic sinusoidal endothelial cells infected with HCMV possess the capacity to modulate allogeneic T cell recruitment and activation, thereby providing a plausible mechanism of how HCMV infection is able to enhance hepatic immune activation. METHODS: Human hepatic sinusoidal endothelial cells were isolated from explanted livers and infected with recombinant endotheliotropic HCMV. We used static and flow-based models to quantify adhesion and transendothelial migration of allogeneic T cell subsets and determine their post-migratory phenotype and function. RESULTS: HCMV infection of primary human hepatic sinusoidal endothelial cells facilitated ICAM-1 and CXCL10-dependent CD4 T cell transendothelial migration under physiological levels of shear stress. Recruited T cells were primarily non-virus-specific CXCR3(hi) effector memory T cells, which demonstrated features of LFA3-dependent Th1 activation after migration, and activated regulatory T cells, which retained a suppressive phenotype following transmigration. CONCLUSIONS: The ability of infected hepatic endothelium to recruit distinct functional CD4 T cell subsets shows how HCMV facilitates hepatic inflammation and immune activation and may simultaneously favor virus persistence.

Contact-dependent depletion of hydrogen peroxide by catalase is a novel mechanism of myeloid-derived suppressor cell induction operating in human hepatic stellate cells.

Myeloid-derived suppressor cells (MDSC) represent a unique cell population with distinct immunosuppressive properties that have been demonstrated to shape the outcome of malignant diseases. Recently, human hepatic stellate cells (HSC) have been reported to induce monocytic-MDSC from mature CD14(+) monocytes in a contact-dependent manner. We now report a novel and unexpected mechanism by which CD14(+)HLADR(low/-) suppressive cells are induced by catalase-mediated depletion of hydrogen peroxide (H2O2). Incubation of CD14(+) monocytes with catalase led to a significant induction of functional MDSC compared with media alone, and H2O2 levels inversely correlated with MDSC frequency (r = -0.6555, p < 0.05). Catalase was detected in primary HSC and a stromal cell line, and addition of the competitive catalase inhibitor hydroxylamine resulted in a dose-dependent impairment of MDSC induction and concomitant increase of H2O2 levels. The NADPH-oxidase subunit gp91 was significantly increased in catalase-induced MDSC as determined by quantitative PCR outlining the importance of oxidative burst for the induction of MDSC. These findings represent a so far unrecognized link between immunosuppression by MDSC and metabolism. Moreover, this mechanism potentially explains how stromal cells can induce a favorable immunological microenvironment in the context of tissue oxidative stress such as occurs during cancer therapy.

Elevated miR-122 serum levels are an independent marker of liver injury in inflammatory diseases.

BACKGROUND & AIMS: Serum concentrations of miR-122 were proposed as a marker for various inflammatory diseases, but the mechanisms driving alterations in miR-122 serum levels are unknown. METHODS: We analysed miR-122 serum levels and hepatic miR-122 expression in mice after hepatic ischaemia and reperfusion (I/R) injury. These data were compared with data from mice after caecal pole ligation and puncture (CLP) procedure. To translate these data into the human, we analysed miR-122 serum concentrations in a cohort of 223 patients with critical illness and 57 patients with cirrhosis. RESULTS: We detected strongly elevated levels of miR-122 in mice after hepatic I/R injury. miR-122-concentrations correlated with the degree of liver damage according to AST/ALT and were associated with the presence of hepatic cell death detected by TUNEL staining. miR-122 levels were elevated in the cellular supernatants in an in vitro model of hepatocyte injury, supporting the hypothesis that the passive release of miR-122 represents a surrogate for hepatocyte death in liver injury. Moreover, miR-122 levels were almost normal in patients with cirrhosis without ongoing liver damage, but were elevated when liver injury was present. In contrast to previous assumptions, miR-122-concentrations were independent of the presence of infection/sepsis in mice or human patients. miR-122 levels did not correlate with disease severity or mortality in critically ill patients. In contrast, serum miR-122 levels strictly correlated with the presence of hepatic injury in these patients. CONCLUSION: In mice and humans, miR-122 levels represent an independent and potent marker of ongoing liver injury and hepatic cell death regardless of the underlying disease.

CCR1 and CCR2 antagonists.

Chemokines constitute a family of small heparin-binding proteins which orchestrate the infiltration of leukocytes during inflammation, but also directly influence other physiological and pathophysiological processes. In humans, more than 40 chemokines are known binding to around 18 G-protein-coupled receptors. A non-redundant role of certain chemokines and their receptors has been identified within the last years in inflammation and host defense. Among chemokine receptors, the CC chemokine receptors CCR1 and CCR2 have been shown to play a crucial role in these processes. Importantly, these receptors have already been targeted by specific antagonists in early human trials for autoimmune and infectious diseases. Although most of these antagonists failed to show any significant efficacy in the clinic, the knowledge of their biological effects could henceforth offer new avenues with optimal strategies for producing successful therapeutics.

Clinical relevance and cellular source of elevated soluble urokinase plasminogen activator receptor (suPAR) in acute liver failure.

BACKGROUND & AIMS: Acute liver failure (ALF) is a life-threatening condition with a high mortality rate. The expression of urokinase plasminogen activator receptor (uPAR, CD87) and release of its shedded receptor into serum as soluble uPAR (suPAR) have been closely related to immune activation and prognosis in systemic inflammation and cirrhosis. We now aimed at investigating the clinical relevance and cellular source of uPAR and circulating suPAR in ALF. METHODS: Serum suPAR concentrations were measured in 48 ALF patients and 62 healthy controls from a German liver transplantation centre. Hepatic immune cell subsets and uPAR expression were studied by FACS, qPCR and immunohistochemistry. RESULTS: Circulating suPAR levels were significantly increased in ALF patients, independent from the underlying aetiology, in comparison to controls. Serum suPAR concentrations were closely correlated with parameters reflecting liver cell injury, decreased liver function and the model of end-stage liver disease (MELD) score in ALF patients. By immunohistochemistry from explanted livers, ALF was associated with distinct immune cell accumulation and strong up-regulation of intrahepatic uPAR mRNA expression. CD87 (uPAR) expression was specifically detected on intrahepatic 'non-classical' monocytes (CD14(+) CD16(+) ), NKT and CD56(dim) NK cells isolated from human liver, but not on parenchymal or other non-parenchymal hepatic cell types. Membrane-bound uPAR was rapidly cleaved from monocytes upon inflammatory stimulation by lipopolysaccharide (LPS) and partially by co-cultured lymphocytes. CONCLUSIONS: Similar to its prognostic properties in patients with sepsis or cirrhosis, intrahepatic uPAR activation and serum suPAR concentrations might serve as an interesting biomarker in ALF.