Circulating CXCL10 in cirrhotic portal hypertension might reflect systemic inflammation and predict ACLF and mortality.

BACKGROUND & AIMS: CXCR% ligands play an important role in hepatic injury, inflammation and fibrosis. While CXCL9 and CXCL11 are associated with survival in patients receiving transjugular intrahepatic portosystemic shunt (TIPS), the role of CXCL10 in severe portal hypertension remains unknown. METHODS: A total of 89 cirrhotic patients were analysed. CXCL10 protein levels were measured in portal and hepatic blood at TIPS insertion and 2 weeks later in 24 patients. CXCL10 and IL8 levels were assessed in portal, hepatic, cubital vein and right atrium blood in a further 25 patients at TIPS insertion. Furthermore, real-time PCR determined hepatic CXCL10-mRNA in 40 cirrhotic patients. RESULTS: Hepatic CXCL10 showed no association with decompensation. By contrast, circulating CXCL10-levels were higher in portal than in hepatic vein blood, suggesting an extrahepatic source of CXCL10 in cirrhosis. However, CXCL10 protein in blood samples from portal, hepatic, cubital veins and right atrium correlated excellently with each other and with IL-8 levels. Higher CXCL10 circulating levels were associated with presence of ascites and higher Child scores. Higher CXCL10 circulating protein levels were associated with acute decompensation, acute-on-chronic liver failure (ACLF) and independently with mortality. Moreover, a decrease in CXCL10 protein levels after TIPS insertion was associated with better survival in each cohort and analysed together. DISCUSSION: Circulating CXCL10 possibly reflects systemic inflammation and it is correlated with acute decompensation, ACLF and complications in patients with severe portal hypertension receiving TIPS. CXCL10 predicts survival in these patients and a decrease in CXCL10 after TIPS may be considered a good prognostic factor.

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.

Protective role of macrophage migration inhibitory factor in nonalcoholic steatohepatitis.

MIF is an inflammatory cytokine but is hepatoprotective in models of hepatotoxin-induced liver fibrosis. Hepatic fibrosis can also develop from metabolic liver disease, such as nonalcoholic fatty liver disease (NASH). We investigated the role of MIF in high-fat or methionine- and choline-deficient diet mouse models of NASH. Mif(-/-) mice showed elevated liver triglyceride levels (WT, 53±14 mg/g liver; Mif(-/-), 103±7 mg/g liver; P<0.05) and a 2-3-fold increased expression of lipogenic genes. Increased fatty degeneration in the livers of Mif(-/-) mice was associated with increased hepatic inflammatory cells (1.6-fold increase in F4/80(+) macrophages) and proinflammatory cytokines (e.g., 2.3-fold increase in Tnf-α and 2-fold increase in Il-6 expression). However, inflammatory cells and cytokines were decreased by 50-90% in white adipose tissue (WAT) of Mif(-/-) mice. Subset analysis showed that macrophage phenotypes in livers of Mif(-/-) mice were skewed toward M2 (e.g., 1.7-fold and 2.5-fold increase in Arg1 and Il-13, respectively, and 2.5-fold decrease in iNos), whereas macrophages were generally reduced in WAT of these mice (70% reduction in mRNA expression of F4/80(+) macrophages). The protective MIF effect was scrutinized in isolated hepatocytes. MIF reversed inflammation-induced triglyceride accumulation in Hepa1-6 cells and primary hepatocytes and also attenuated oleic acid-elicited triglyceride increase in 3T3-L1 adipocytes. Protection from fatty hepatocyte degeneration was paralleled by a 2- to 3-fold reduction by MIF of hepatocyte proinflammatory cytokine production. Blockade of MIF receptor cluster of differentiation 74 (CD74) but not of CXCR2 or CXCR4 fully reverted the protective effect of MIF, comparable to AMPK inhibition. In summary, we demonstrate that MIF mediates hepatoprotection through the CD74/AMPK pathway in hepatocytes in metabolic models of liver injury.

Chemokines in tissue fibrosis.

Fibrosis or scarring of diverse organs and tissues is considered as a pathologic consequence of a chronically altered wound healing response which is tightly linked to inflammation and angiogenesis. The recruitment of immune cells, local proliferation of fibroblasts and the consecutive accumulation of extracellular matrix proteins are common pathophysiological hallmarks of tissue fibrosis, irrespective of the organ involved. Chemokines, a family of chemotactic cytokines, appear to be central mediators of the initiation as well as progression of these biological processes. Traditionally chemokines have only been considered to play a critical role in orchestrating the influx of immune cells to sites of tissue injury. However, within the last years, further aspects of chemokine biology including fibroblast activation and angiogenesis have been deciphered in tissue fibrosis of many different organs. Interestingly, certain chemokines appear to mediate common effects in liver, kidney, lung, and skin of various animal models, while others mediate tissue specific effects. These aspects have to be kept in mind when extrapolating data of animal studies to early human trials. Nevertheless, the further understanding of chemokine effects in tissue fibrosis might be an attractive approach for identifying novel therapeutic targets in chronic organ damage associated with high morbidity and mortality. This article is part of a Special Issue entitled: Fibrosis: Translation of basic research to human disease.

Platelet-derived growth factor (PDGF)-C neutralization reveals differential roles of PDGF receptors in liver and kidney fibrosis.

Platelet-derived growth factors (PDGF) are key mediators of organ fibrosis. We investigated whether PDGF-C(-/-) mice or mice treated with neutralizing PDGF-C antibodies are protected from bile duct ligation-induced liver fibrosis, and we compared the effects with those of PDGF-C deficiency or neutralization on kidney fibrosis induced by unilateral ureteral obstruction. Unexpectedly, and in contrast to kidney fibrosis, PDGF-C deficiency or antagonism did not protect from liver fibrosis or functional liver impairment. Furthermore, the hepatic infiltration of monocytes/macrophages/dendritic cells and chemokine mRNA expression (CC chemokine ligand [CCL]5, CCL2, and CC chemokine receptor 2 [CCR2]) remained unchanged. Transcript expression of PDGF ligands increased in both liver and kidney fibrosis and was not affected by neutralization of PDGF-C. In kidney fibrosis, PDGF-C deficiency or antagonism led to reduced expression and signaling of PDGF-receptor (R)-α- and PDGFR-ß-chains. In contrast, in liver fibrosis there was either no difference (PDGF-C(-/-) mice) or even an upregulation of PDGFR-ß and signaling (anti-PDGF-C group). Finally, in vitro studies in portal myofibroblasts pointed to a predominant role of PDGF-B and PDGF-D signaling in liver fibrosis. In conclusion, our study revealed significant differences between kidney and liver fibrosis in that PDGF-C mediates kidney fibrosis, whereas antagonism of PDGF-C in liver fibrosis appears to be counteracted by significant upregulation and increased PDGFR-ß signaling. PDGF-C antagonism, therefore, may not be effective to treat liver fibrosis.

Proapoptotic effects of the chemokine, CXCL 10 are mediated by the noncognate receptor TLR4 in hepatocytes.

UNLABELLED: Aberrant expression of the chemokine CXC chemokine ligand (CXCL)10 has been linked to the severity of hepatitis C virus (HCV)-induced liver injury, but the underlying molecular mechanisms remain unclear. In this study, we describe a yet-unknown proapoptotic effect of CXCL10 in hepatocytes, which is not mediated through its cognate chemokine receptor, but the lipopolysaccharide receptor Toll-like receptor 4 (TLR4). To this end, we investigated the link of CXCL10 expression with apoptosis in HCV-infected patients and in murine liver injury models. Mice were treated with CXCL10 or neutralizing antibody to systematically analyze effects on hepatocellular apoptosis in vivo. Direct proapoptotic functions of CXCL10 on different liver cell types were evaluated in detail in vitro. The results showed that CXCL10 expression was positively correlated with liver cell apoptosis in humans and mice. Neutralization of CXCL10 ameliorated concanavalin A-induced tissue injury in vivo, which was strongly associated with reduced liver cell apoptosis. In vitro, CXCL10 mediated the apoptosis of hepatocytes involving TLR4, but not CXC chemokine receptor 3 signaling. Specifically, CXCL10 induced long-term protein kinase B and Jun N-terminal kinase activation, leading to hepatocyte apoptosis by caspase-8, caspase-3, and p21-activated kinase 2 cleavage. Accordingly, systemic application of CXCL10 led to TLR4-induced liver cell apoptosis in vivo. CONCLUSION: The results identify CXCL10 and its noncognate receptor, TLR4, as a proapoptotic signaling cascade during liver injury. Antagonism of the CXCL10/TLR4 pathway might be a therapeutic option in liver diseases associated with increased apoptosis.

Macrophage migration inhibitory factor (MIF) exerts antifibrotic effects in experimental liver fibrosis via CD74.

Macrophage migration inhibitory factor (MIF) is a pleiotropic inflammatory cytokine that has been implicated in various inflammatory diseases. Chronic inflammation is a mainstay of liver fibrosis, a leading cause of morbidity worldwide, but the role of MIF in liver scarring has not yet been elucidated. Here we have uncovered an unexpected antifibrotic role for MIF. Mice genetically deleted in Mif (Mif(-/-)) showed strongly increased fibrosis in two models of chronic liver injury. Pronounced liver fibrosis in Mif(-/-) mice was associated with alterations in fibrosis-relevant genes, but not by a changed intrahepatic immune cell infiltration. Next, a direct impact of MIF on hepatic stellate cells (HSC) was assessed in vitro. Although MIF alone had only marginal effects on HSCs, it markedly inhibited PDGF-induced migration and proliferation of these cells. The inhibitory effects of MIF were mediated by CD74, which we detected as the most abundant known MIF receptor on HSCs. MIF promoted the phosphorylation of AMP-activated protein kinase (AMPK) in a CD74-dependent manner and, in turn, inhibition of AMPK reversed the inhibition of PDGF-induced HSC activation by MIF. The pivotal role of CD74 in MIF-mediated antifibrotic properties was further supported by augmented liver scarring of Cd74(-/-) mice. Moreover, mice treated with recombinant MIF displayed a reduced fibrogenic response in vivo. In conclusion, we describe a previously unexplored antifibrotic function of MIF that is mediated by the CD74/AMPK signaling pathway in HSCs. The results imply MIF and CD74 as targets for treatment of liver diseases.

Complement factor 5 is a quantitative trait gene that modifies liver fibrogenesis in mice and humans.

Fibrogenesis or scarring of the liver is a common consequence of all chronic liver diseases. Here we refine a quantitative trait locus that confers susceptibility to hepatic fibrosis by in silico mapping and show, using congenic mice and transgenesis with recombined artificial chromosomes, that the gene Hc (encoding complement factor C5) underlies this locus. Small molecule inhibitors of the C5a receptor had antifibrotic effects in vivo, and common haplotype-tagging polymorphisms of the human gene C5 were associated with advanced fibrosis in chronic hepatitis C virus infection. Thus, the mouse quantitative trait gene led to the identification of an unknown gene underlying human susceptibility to liver fibrosis, supporting the idea that C5 has a causal role in fibrogenesis across species.

Chemokine Cxcl9 attenuates liver fibrosis-associated angiogenesis in mice.

UNLABELLED: Recent data suggest that the chemokine receptor CXCR3 is functionally involved in fibroproliferative disorders, including liver fibrosis. Neoangiogenesis is an important pathophysiological feature of liver scarring, but a functional role of angiostatic CXCR3 chemokines in this process is unclear. We therefore investigated neoangiogenesis in carbon tetrachloride (CCl(4))-induced liver fibrosis in Cxcr3(-/-) and wildtype mice by histological, molecular, and functional imaging methods. Furthermore, we assessed the direct role of vascular endothelial growth factor (VEGF) overexpression on liver angiogenesis and the fibroproliferative response using a Tet-inducible bitransgenic mouse model. The feasibility of attenuation of angiogenesis and associated liver fibrosis by therapeutic treatment with the angiostatic chemokine Cxcl9 was systematically analyzed in vitro and in vivo. The results demonstrate that fibrosis progression in Cxcr3(-/-) mice was strongly linked to enhanced neoangiogenesis and VEGF/VEGFR2 expression compared with wildtype littermates. Systemic VEGF overexpression led to a fibrogenic response within the liver and was associated with a significantly increased Cxcl9 expression. In vitro, Cxcl9 displayed strong antiproliferative and antimigratory effects on VEGF-stimulated endothelial cells and stellate cells by way of reduced VEGFR2 (KDR), phospholipase Cγ (PLCγ), and extracellular signal-regulated kinase (ERK) phosphorylation, identifying this chemokine as a direct counter-regulatory molecule of VEGF signaling within the liver. Accordingly, systemic administration of Cxcl9 led to a strong attenuation of neoangiogenesis and experimental liver fibrosis in vivo. CONCLUSION: The results identify direct angiostatic and antifibrotic effects of the Cxcr3 ligand Cxcl9 in a model of experimental liver fibrosis. The amelioration of liver damage by systemic application of Cxcl9 might offer a novel therapeutic approach for chronic liver diseases associated with increased neoangiogenesis.

BASIC--a bile acid-sensitive ion channel highly expressed in bile ducts.

Brain liver intestine Na+ channel (BLINaC) is an ion channel of the DEG/ENaC gene family of unknown function. BLINaC from rats (rBLINaC) and humans (INaC) is inactive at rest, and its mode of activation has remained unclear. Here, we show that the BLINaC protein localizes to cholangiocytes, epithelial cells that line bile ducts. Moreover, we provide evidence that rBLINaC and INaC are robustly activated by bile acids, in particular chenodeoxycholic acid and hyodeoxycholic acid (EC50=2.1±0.05 mM). Thus, BLINaC appears to be an epithelial cation channel of bile ducts sensitive to physiological concentrations of bile acids. BLINaC is related to acid-sensing ion channels (ASICs) and to the epithelial Na+ channel (ENaC) and shares ligand activation with ASICs and epithelial localization with ENaC. Therefore, based on the close homology of BLINaC to ASICs and its activation by bile acids, we propose to rename BLINaC bile acid-sensitive ion channel (BASIC).

Hepatocyte growth factor/c-Met signalling is important for the selection of transplanted hepatocytes.

BACKGROUND: At present hepatocyte transplantation is a promising option for cellular therapy of end-stage liver diseases. However, the underlying molecular mechanisms need to be better defined in order to translate this technique into clinical use. This study investigated the cursiv relevance of hepatocyte growth factor (HGF)/c-Met signalling for hepatocyte repopulation after transplantion. METHODS: Wild-type mice (c-Met(loxP/loxP)) and hepatocyte-specific conditional c-Met (HGF receptor) knockout (c-Met(Δhepa)) mice were used as donors and recipients for hepatocyte transplantation. RESULTS: Transplantation experiments revealed two major findings. First it was demonstrated that c-Met is indispensable in donor cells, as c-Met(Δhepa) cells did not repopulate recipient livers after transplantation. Second, genetic deletion of c-Met in recipient hepatocytes resulted in enhanced expansion of unmodified donor cells in host livers (up to 250-fold after 12 weeks). The relevant mechanisms for this observation in c-Met(Δhepa) host hepatocytes could be defined. c-Met(Δhepa) hepatocytes showed enhanced apoptosis, reduced cellular proliferation and a lack of AKT-kinase and STAT3 activation. In addition, tissue remodelling was changed in c-Met(Δhepa) recipient livers. Therefore, the lack of pro-proliferative transcription factors, increased apoptosis and changes in matrix-remodelling inhibit host cell proliferation in c-Met(Δhepa) recipient livers and thus favour repopulation of transplanted hepatocytes. Therapeutically liver repopulation could be increased through adenoviral expression of NK-4--an inhibitor of HGF signalling--in host hepatocytes. CONCLUSION: HGF/c-Met plays a crucial role in host and donor cells of the liver for the cursiv selection of transplanted hepatocytes. Modulating HGF-dependent signalling seems a promising therapeutic option to favour expansion of transplanted hepatocytes.

Adaptive immunity suppresses formation and progression of diethylnitrosamine-induced liver cancer.

BACKGROUND: Hepatocellular carcinoma (HCC) is a typical inflammation-associated cancer, but may also provoke antitumour immune responses whose significance and underlying mechanisms are incompletely understood. OBJECTIVE: To characterise immune responses in the diethylnitrosamine (DEN)-liver cancer mouse model. DESIGN: Tumour development and immune cell functions upon DEN treatment were compared between C57BL/6 wild-type (WT), chemokine scavenging receptor D6-deficient, B cell- (Igh6), CD4 T cell- (MHC-II) and T-/B cell-deficient (Rag1) mice. Relevance for human HCC was tested by comparing gene array results from 139 HCC tissues. RESULTS: The induction of premalignant lesions after 24 weeks and of HCC-like tumours after 42 weeks by DEN in mice was accompanied by significant leucocyte infiltration in the liver and upregulation of distinct intrahepatic chemokines (CCL2, CCL5, CXCL9). Macrophages and CD8 (cytotoxic) T cells were most prominently enriched in tumour-bearing livers, similar to samples from human HCC. Myeloid-derived suppressor cells (MDSC) increased in extrahepatic compartments of DEN-treated mice (bone marrow, spleen). The contribution of immune cell subsets for DEN-induced hepatocarcinogenesis was functionally dissected. In D6(-/-) mice, which lack the chemokine scavenging receptor D6, hepatic macrophage infiltration was significantly increased, but tumour formation and progression did not differ from that of WT mice. In contrast, progression of hepatic tumours (numbers, diameters, tumour load) was strikingly enhanced in T-/B cell-deficient Rag1(-/-) mice upon DEN treatment. When mice deficient for B cells (Igh6(-/-), µMT) or major histocompatibility complex II were used, the data indicated that T cells prevent initial tumour formation, while B cells critically limit growth of established tumours. Accordingly, in tumour-bearing mice antibody production against liver-related model antigen was enhanced, indicating tumour-associated B cell activation. In agreement, T and B cell pathways were differentially regulated in gene array analyses from 139 human HCC tissues and significantly associated with patients' survival. CONCLUSIONS: Distinct axes of the adaptive immune system, which are also prognostic in human HCC, actively suppress DEN-induced hepatocarcinogenesis by controlling tumour formation and progression.

The chemokine receptor CXCR3 limits injury after acute toxic liver damage.

Although acute liver failure is a rare disease, its presence is associated with high morbidity and mortality in affected patients. While a contribution of the immune system to the outcome of toxic liver failure is anticipated, functionally relevant immune cell receptors for liver cell damage need to be better defined. We here investigate the relevance of the chemokine receptor CXCR3, which is important for hepatic immune cell infiltration, in a model of experimental acute liver failure. Liver injury was induced by a single intraperitoneal injection of carbon tetrachloride (CCl(4)) in CXCR3(-/-), CCR1(-/-), CCR5(-/-) and wild-type mice. In this model, CXCR3(-/-) mice displayed augmented liver damage compared with all other mouse strains as assessed by liver histology and serum transaminases 24 and 72 h after injury. Phenotypically, CXCR3(-/-) mice had significantly reduced intrahepatic NK and NKT cells after injury at all investigated time points (all P<0.05), but strongly elevated expression levels of IL1-ß, TNF-α and IFN-γ. In line with a functional role of innate immune cells, wild-type mice depleted for NK cells with an anti-ASIALO GM1 antibody before liver injury also displayed increased liver injury after CCl(4) challenge. CXCR3(-/-) and NK cell-depleted mice show reduced apoptotic liver cells (TUNEL assay), but more necrotic hepatocytes. Functionally, the augmented liver cell necrosis in CXCR3(-/-) and NK cell-depleted mice was associated with increased expression of high mobility group 1 (HMGB1) protein and a consecutive enhanced infiltration of neutrophils into the liver. In conclusion, the results demonstrate a primarily unexpected beneficial role of CXCR3 in acute toxic liver injury. These findings should be taken into account when planning trials with CXCR3 antagonists.

A 7-gene signature of the recipient predicts the progression of fibrosis after liver transplantation for hepatitis C virus infection.

Fibrosis recurrence after liver transplantation (LT) for hepatitis C virus (HCV) is a universal event and strongly determines a patient's prognosis. The recipient risk factors for fibrosis recurrence are still poorly defined. Here we assess a genetic risk score as a predictor of fibrosis after LT. The cirrhosis risk score (CRS), which comprises allele variants in 7 genes (adaptor-related protein complex 3 S2, aquaporin 2, antizyme inhibitor 1, degenerative spermatocyte homolog 1 lipid desaturase, syntaxin binding protein 5-like, toll-like receptor 4, and transient receptor potential cation channel M5), was calculated for 137 patients who underwent LT for HCV infection and experienced HCV reinfection of the graft. The patients were stratified into 3 CRS categories: <0.5, 0.5 to 0.7, and >0.7. All patients underwent protocol biopsy after LT (median follow-up = 5 years), and liver fibrosis was assessed according to the Desmet and Scheuer score. The data were analyzed with univariate and multivariate analyses. The results showed that the highest CRS category was strongly associated with the presence of F2 or F3 fibrosis in protocol biopsy samples 1, 3, and 5 years after LT (P = 0.006, P = 0.001, and P = 0.02, respectively). Overall, 75.0% of the patients with a CRS > 0.7 developed at least F2 fibrosis, whereas 51.5% developed F3 fibrosis during follow-up. The predictive value of the CRS for fibrosis progression was independent of known clinical risk factors, including the age of the donor, the sex of the recipient, and the occurrence of acute rejection. A Kaplan-Meier analysis confirmed the prognostic value of the CRS with respect to the recurrence of severe liver fibrosis in HCV-infected patients after LT (log rank = 6.23, P = 0.03). In conclusion, the genetic signature of the recipient predicts the likelihood of severe liver fibrosis in the graft after HCV recurrence. The CRS might help with early clinical decision making (eg, the selection of patients for antiviral therapy after LT).

Serum chemokine CXC ligand 10 (CXCL10) predicts fibrosis progression after liver transplantation for hepatitis C infection.

UNLABELLED: The recurrence of liver fibrosis after liver transplantation (LT) for hepatitis C virus (HCV) infection is responsible for graft loss and patient mortality. Although the contribution of the immune system to fibrosis recurrence is anticipated, systematic studies evaluating immune parameters as predictive markers of allograft fibrosis are lacking. The infiltration of immune cells into the graft is governed by chemokines. Here we assessed the predictive value of serum levels of chemokines [chemokine (C-X-C motif) ligand 9 (CXCL9), CXCL10, CXCL11, and chemokine (C-C motif) ligand 2 (CCL2)] with respect to fibrosis recurrence after LT in 90 HCV-infected organ recipients. Chemokines were determined within the first and third years after LT and were correlated with histological fibrosis progression in protocol biopsy samples at 1, 3, 5, and 7 years (median follow-up = 3 years). The association of chemokines with fibrosis progression was assessed by univariate and multivariate analyses and by Cox regression analysis. The results for the analyzed chemokines showed that CXCL10 levels in the first year after LT were strongly associated with early fibrosis recurrence (P = 0.005) independently of risk confounders (including the donor age, HCV viral load, HCV genotype, acute rejection, and inflammatory activity). As assessed by Cox regression analysis, a CXCL10 serum level ≤ 140 pg/mL was significantly predictive of the absence of F2 fibrosis (P = 0.001), whereas a level ≤ 220 pg/mL early after LT predicted the absence of F3 fibrosis during follow-up (P = 0.035). CONCLUSION: CXCL10 is an independent biomarker of the recurrence of significant fibrosis after LT for HCV infection. These results might guide patients' care after transplantation and help us to select optimal candidates for antiviral therapy post-LT.

Soluble urokinase plasminogen activator receptor is associated with progressive liver fibrosis in hepatitis C infection.

BACKGROUND: Progressive liver fibrosis is the main predictor of disease outcome in chronic hepatitis C viral (HCV) infection. Although the importance of the coagulation cascade has been suggested in liver fibrogenesis, the role of the fibrinolytic pathway is yet unclear. GOAL: We evaluated the association of serum levels of the fibrinolysis-associated soluble urokinase plasminogen activator receptor (suPAR) with the severity of liver fibrosis in HCV infection. STUDY: suPAR serum levels were assessed in 146 chronically HCV-infected patients of 2 independent cohorts (64 subjects in the screening cohort, 82 in the validation cohort) by enzyme-linked immunosorbent assay and correlated with biopsy-proven histologic stage of liver fibrosis and noninvasive liver fibrosis markers (aspartate transaminase to platelets ratio index score, transient elastography). RESULTS: suPAR serum levels were strongly associated with the histologic stage of liver fibrosis in both cohorts (P<0.0001). Although mean suPAR levels in patients with F1 and F2 fibrosis were not different from healthy control subjects, they were significantly increased at higher stages of liver fibrosis (F3 and F4, P<0.0001). suPAR values had a high diagnostic specificity and sensitivity to differentiate mild/moderate fibrosis (F1/F2) from severe fibrosis (F3/F4) with an area under curve of 0.774 (P=0.0001) and for the differentiation of noncirrhosis from cirrhosis (F1/F2/F3 vs. F4, area under curve 0.791, P=0.0001). SuPAR serum levels were also strongly correlated to the noninvasive fibrosis markers aspartate transaminase to platelets ratio index score (r=0.52) and transient elastography (r=0.44, both P<0.0001). CONCLUSIONS: Serum suPAR levels were robust markers of liver fibrosis in 2 cohorts with a comparable diagnostic accuracy for prediction of severe liver fibrosis as established noninvasive marker.

The fractalkine receptor CX3CR1 protects against liver fibrosis by controlling differentiation and survival of infiltrating hepatic monocytes.

UNLABELLED: Chemokines modulate inflammatory responses that are prerequisites for organ fibrosis upon liver injury. Monocyte-derived hepatic macrophages are critical for the development, maintenance, and resolution of hepatic fibrosis. The specific role of monocyte-associated chemokine (C-X3-C motif) receptor 1 (CX3CR1) and its cognate ligand fractalkine [chemokine (C-X3-C motif) ligand 1)] in liver inflammation and fibrosis is currently unknown. We examined 169 patients with chronic liver diseases and 84 healthy controls; we found that CX3CL1 is significantly up-regulated in the circulation upon disease progression, whereas CX3CR1 is down-regulated intrahepatically in patients with advanced liver fibrosis or cirrhosis. To analyze the functional relevance of this pathway, two models of experimental liver fibrosis were applied to wild-type (WT) and CX3CR1-deficient mice. Fractalkine expression was induced upon liver injury in mice, primarily in hepatocytes and hepatic stellate cells. CX3CR1(-/-) animals developed greater hepatic fibrosis than WT animals with carbon tetrachloride-induced and bile duct ligation-induced fibrosis. CX3CR1(-/-) mice displayed significantly increased numbers of monocyte-derived macrophages within the injured liver. Chimeric animals that underwent bone marrow transplantation revealed that CX3CR1 restricts hepatic fibrosis progression and monocyte accumulation through mechanisms exerted by infiltrating immune cells. In the absence of CX3CR1, intrahepatic monocytes develop preferentially into proinflammatory tumor necrosis factor-producing and inducible nitric oxide synthase-producing macrophages. CX3CR1 represents an essential survival signal for hepatic monocyte-derived macrophages by activating antiapoptotic bcl2 expression. Monocytes/macrophages lacking CX3CR1 undergo increased cell death after liver injury, which then perpetuates inflammation, promotes prolonged inflammatory monocyte infiltration into the liver, and results in enhanced liver fibrosis. CONCLUSION: CX3CR1 limits liver fibrosis in vivo by controlling the differentiation and survival of intrahepatic monocytes. The opposing regulation of CX3CR1 and fractalkine in patients suggests that pharmacological augmentation of this pathway may represent a possible therapeutic antifibrotic strategy.

Lack of interleukin-6/glycoprotein 130/signal transducers and activators of transcription-3 signaling in hepatocytes predisposes to liver steatosis and injury in mice.

UNLABELLED: A deregulated cytokine balance is involved in triggering the sequence from steatosis to nonalcoholic steatohepatitis, ultimately leading to liver fibrosis and cancer. To better define the role of proinflammatory interleukin-6 (IL-6)-type cytokines in hepatocytes we investigated the role of IL-6 and its shared receptor, glycoprotein 130 (gp130), in a mouse model of steatohepatitis. IL-6(-/-) mice were fed a choline-deficient, ethionine-supplemented (CDE) diet. Conditional gp130 knockout and knockin mice were used to achieve hepatocyte-specific deletion of gp130 (gp130(Deltahepa)), gp130-dependent rat sarcoma (Ras)-(gp130(DeltahepaRas)), and signal transducers and activators of transcription (STAT)-(gp130(DeltahepaSTAT)) activation. CDE-treated IL-6(-/-) mice showed a significant hepatic steatosis at 2 weeks after feeding. The mice rapidly developed elevated fasting blood glucose, insulin serum levels, and transaminases. To better define IL-6-dependent intracellular pathways, specifically in hepatocytes, we next treated gp130(Deltahepa) mice with a CDE diet. These animals also developed a marked steatosis with hyperglycemia and displayed elevated insulin serum levels. Additionally, gp130(Deltahepa) animals showed an imbalanced inflammatory response with increased hepatic tumor necrosis factor-alpha and decreased adiponectin messenger RNA levels. Dissecting the hepatocyte-specific gp130-dependent pathways revealed a similar disease phenotype in gp130(DeltahepaSTAT) mice, whereas gp130(DeltahepaRas) animals were protected. In CDE-treated mice lack of gp130-STAT3 signaling was associated with immune-cell-infiltration, jun kinase-activation, a blunted acute-phase-response, and elevated transaminases. Furthermore, gp130(Deltahepa) and gp130(DeltahepaSTAT) mice showed beginning signs of liver fibrosis compared to gp130(DeltahepaRas) mice and controls. CONCLUSION: During CDE treatment mice lacking IL-6 and gp130-STAT signaling in hepatocytes are prone to hepatic metabolic changes and inflammation. This ultimately leads to progressive steatohepatitis with signs of liver remodeling. Thus, the presented model allows one to further dissect the role of IL-6/gp130-type signaling in hepatocytes during fatty liver degeneration to define new therapeutic targets in metabolic liver diseases.

CXC chemokine ligand 4 (Cxcl4) is a platelet-derived mediator of experimental liver fibrosis.

UNLABELLED: Liver fibrosis is a major cause of morbidity and mortality worldwide. Platelets are involved in liver damage, but the underlying molecular mechanisms remain elusive. Here, we investigate the platelet-derived chemokine (C-X-C motif) ligand 4 (CXCL4) as a molecular mediator of fibrotic liver damage. Serum concentrations and intrahepatic messenger RNA of CXCL4 were measured in patients with chronic liver diseases and mice after toxic liver injury. Platelet aggregation in early fibrosis was determined by electron microscopy in patients and by immunohistochemistry in mice. Cxcl4(-/-) and wild-type mice were subjected to two models of chronic liver injury (CCl(4) and thioacetamide). The fibrotic phenotype was analyzed by histological, biochemical, and molecular analyses. Intrahepatic infiltration of immune cells was investigated by fluorescence-activated cell sorting, and stellate cells were stimulated with recombinant Cxcl4 in vitro. The results showed that patients with advanced hepatitis C virus-induced fibrosis or nonalcoholic steatohepatitis had increased serum levels and intrahepatic CXCL4 messenger RNA concentrations. Platelets were found directly adjacent to collagen fibrils. The CCl(4) and thioacetamide treatment led to an increase of hepatic Cxcl4 levels, platelet activation, and aggregation in early fibrosis in mice. Accordingly, genetic deletion of Cxcl4 in mice significantly reduced histological and biochemical liver damage in vivo, which was accompanied by changes in the expression of fibrosis-related genes (Timp-1 [tissue inhibitor of matrix metalloproteinase 1], Mmp9 [matrix metalloproteinase 9], Tgf-beta [transforming growth factor beta], IL10 [interleukin 10]). Functionally, Cxcl4(-/-) mice showed a strongly decreased infiltration of neutrophils (Ly6G) and CD8(+) T cells into the liver. In vitro, recombinant murine Cxcl4 stimulated the proliferation, chemotaxis, and chemokine expression of hepatic stellate cells. CONCLUSION: The results underscore an important role of platelets in chronic liver damage and imply a new target for antifibrotic therapies.

Long-term efficacy of tenofovir monotherapy for hepatitis B virus-monoinfected patients after failure of nucleoside/nucleotide analogues.

UNLABELLED: Tenofovir disoproxil fumarate (TDF) has demonstrated high antiviral efficacy in treatment-naive patients with chronic hepatitis B virus (HBV) infection but experience in nucleoside/nucleotide analogue (NA)-experienced patients is limited. In this retrospective multicenter study we therefore assessed the long-term efficacy of TDF monotherapy in patients with prior failure or resistance to different NA treatments. Criteria for inclusion were HBV DNA levels >4.0 log(10) copies/mL at the start and a minimum period of TDF therapy for at least 6 months. In all, 131 patients (mean age 42 +/- 12 years, 95 male, 65% hepatitis B e antigen [HBeAg]-positive) were eligible. Pretreatment consisted of either monotherapy with lamivudine (LAM; n = 18), adefovir (ADV; n = 8), and sequential LAM-ADV therapy (n = 73), or add-on combination therapy with both drugs (n = 29). Three patients had failed entecavir therapy. Resistance analysis in 113 of the 131 patients revealed genotypic LAM and ADV resistance in 62% and 19% of patients, respectively. The mean HBV DNA level at TDF baseline was 7.6 +/- 1.5 log(10) copies/mL. The overall cumulative proportion of patients achieving HBV DNA levels <400 copies/mL was 79% after a mean treatment duration of 23 months (range, 6-60). Although LAM resistance did not influence the antiviral efficacy of TDF, the presence of ADV resistance impaired TDF efficacy (100% versus 52% probability of HBV DNA <400 copies/mL, respectively). However, virologic breakthrough was not observed in any of the patients during the entire observation period. Loss of HBeAg occurred in 24% of patients and HBsAg loss occurred in 3%. No significant adverse events were noticed during TDF monotherapy. CONCLUSION: TDF monotherapy induced a potent and long-lasting antiviral response in NA-experienced patients with previous treatment failure. Our data may have implications for current add-on strategies.