P. Schmitz

Antagonism of the chemokine Ccl5 ameliorates experimental liver fibrosis in mice

Activation of hepatic stellate cells in response to chronic inflammation represents a crucial step in the development of liver fibrosis. However, the molecules involved in the interaction between immune cells and stellate cells remain obscure. Herein, we identify the chemokine CCL5 (also known as RANTES), which is induced in murine and human liver after injury, as a central mediator of this interaction. First, we showed in patients with liver fibrosis that CCL5 haplotypes and intrahepatic CCL5 mRNA expression were associated with severe liver fibrosis. Consistent with this, we detected Ccl5 mRNA and CCL5 protein in 2 mouse models of liver fibrosis, induced by either injection of carbon tetrachloride (CCl4) or feeding on a methionine and choline-deficient (MCD) diet. In these models, Ccl5-/- mice exhibited decreased hepatic fibrosis, with reduced stellate cell activation and immune cell infiltration. Transplantation of Ccl5-deficient bone marrow into WT recipients attenuated liver fibrosis, identifying infiltrating hematopoietic cells as the main source of Ccl5. We then showed that treatment with the CCL5 receptor antagonist Met-CCL5 inhibited cultured stellate cell migration, proliferation, and chemokine and collagen secretion. Importantly, in vivo administration of Met-CCL5 greatly ameliorated liver fibrosis in mice and was able to accelerate fibrosis regression. Our results define a successful therapeutic approach to reduce experimental liver fibrosis by antagonizing Ccl5 receptors.

Antifibrotic Effects of CXCL9 and Its Receptor CXCR3 in Livers of Mice and Humans

BACKGROUND & AIMS Fibrosis is the hallmark of chronic liver diseases, yet many aspects of its mechanism remain to be defined. Chemokines are ubiquitous chemotactic molecules that mediate many acute and chronic inflammatory conditions, and CXC chemokine genes colocalize with a locus previously shown to include fibrogenic genes. We investigated the roles of the chemokine CXCL9 and its receptor CXCR3 in liver fibrosis. METHODS The effects of CXCL variants on fibrogenesis were analyzed using samples from patients with hepatitis C virus infection and by induction of fibrosis in CXCR3(-/-) and wild-type mice. In mice, intrahepatic immune cell subsets were investigated and interferon gamma messenger RNA levels were measured at baseline and after injury. Human serum CXCL9 levels were measured and correlated with CXCL9 variant and fibrosis severity. The effects of stimulation with CXCL9 were investigated on human hepatic stellate cells (LX-2). RESULTS Specific CXCL9 variants were associated with liver fibrosis in mice and humans; CXCL9 serum concentrations correlated with genotypes and levels of fibrosis in patients. In contrast to other chemokines, CXCL9 exerted antifibrotic effects in vitro, suppressing collagen production in LX-2 cells. CXCR3(-/-) mice had increased liver fibrosis; progression was associated with decreased numbers of intrahepatic interferon gamma-positive T cells and reduced interferon gamma messenger RNA, indicating that CXCL9-CXCR3 regulates Th1-associated immune pathways. CONCLUSIONS This is the first description of a chemokine-based antifibrotic pathway in the liver; antifibrotic therapies might be developed to modulate CXC chemokine levels.

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

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 (CCl4 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 CCl4 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‐β [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. (HEPATOLOGY 2010.)

Chemokine Cxcl9 attenuates liver fibrosis‐associated angiogenesis in mice

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 (CCl4)‐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. (HEPATOLOGY 2012)

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.

The chemokine scavenging receptor D6 limits acute toxic liver injury in vivo

Abstract The chemokine decoy receptor D6 is a promiscuous chemokine receptor lacking classical signaling functions. It negatively regulates inflammation by targeting CC chemokines to cellular internalization and degradation. Here we analyze the function of D6 in acute CCl4-induced liver damage in constitutive D6-/- and wild-type mice. The degree of liver injury was assessed by liver histology, serum transaminases, IL-6, and TNFα mRNA expression. Protein levels of D6 ligands (CCL2, CCL3, CCL5) and the non-D6-ligand CXCL9 within the livers were determined by ELISAs. The intrahepatic infiltration of immune cells was characterized by FACS. Genetic deletion of D6 led to prolonged liver damage after acute CCl4 administration. The augmented liver damage in D6-/- mice was associated with increased protein levels of intrahepatic inflammatory chemokines CCL2, CCL3, and CCL5 after 48 h, whereas CXCL9 was not different between knockout and wild-type mice. Functionally, increased intra-hepatic CC chemokine concentrations led to increased infiltration of CD45+ leukocytes, which were mainly identified as T and NK cells. In conclusion, the chemokine scavenger receptor D6 has a non-redundant role in acute toxic liver injury in vivo. These results support the importance of post-translational chemokine regulation and describe a new mechanism of immune modulation within the liver.

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

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. (HEPATOLOGY 2013)

Genetic variations of the chemokine scavenger receptor D6 are associated with liver inflammation in chronic hepatitis C

Chronic hepatitis C (HCV) represents one of the most common chronic infections worldwide and is a major indication for liver transplantation. Liver inflammation is the main predictor of advanced fibrosis in HCV. Inflammatory cells are recruited to the liver by chemokines. Recently, a novel class of chemokine receptors has been characterized that lack signaling functions and are termed scavenger receptors. We determine here whether genetic variations of the scavenger receptor D6 contribute to the grade of liver inflammation in HCV. Four haplotype tagging single nucleotide polymorphisms (SNPs) were identified from HapMap that cover the genetic information of D6 (CCBP2). Among these SNPs, rs4683336 was associated with liver inflammation in qualitative (p = 0.003) and quantitative (p = 0.0086) genotype analysis. This association was confirmed in an independent cohort of HCV-infected patients (p = 0.006 for qualitative and p = 0.0046 for quantitative analysis, respectively). Furthermore, the haplotype that is tagged by marker rs4683336 was significantly correlated with liver inflammation when compared with the most common D6 haplotype (p = 0.014). The importance of genetic variations in D6 was supported through the demonstration of an association of D6 mRNA expression with histologic inflammation in liver biopsies and a considerable range of D6 mRNA expression in isolated human hepatocytes. In conclusion, we demonstrate that variations in a chemokine scavenging receptor are significantly correlated with clinical inflammatory phenotypes such as HCV infection.

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 patients 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). Liver Transpl 18:298–304, 2012.

Interference with Oligomerization and Glycosaminoglycan Binding of the Chemokine CCL5 Improves Experimental Liver Injury

Background The chemokine CCL5 is involved in the recruitment of immune cells and a subsequent activation of hepatic stellate cells (HSC) after liver injury. We here investigate whether inhibition of CCL5 oligomerization and glycosaminoglycan binding by a mutated CCL5 protein (44AANA47-CCL5) has the potential to ameliorate liver cell injury and fibrosis in vivo. Methodology Liver injury was induced in C57BL/6 mice by intraperitoneal injection of carbon tetrachloride (CCl4) in an acute and a chronic liver injury model. Simultaneously, mice received either 44AANA47-CCL5 or vehicle. Liver cell necrosis and fibrosis was analyzed by histology, and measurement of serum transaminases and hydroxyproline. Intrahepatic mRNA expression of fibrosis and inflammation related genes were determined by quantitative RT-PCR and infiltration of immune cells was assessed by FACS analysis and immunocytochemistry. In vitro, HSC were stimulated with conditioned media of T-cell enriched splenocytes. Principal Findings 44AANA47-CCL5 treated mice displayed a significantly reduced degree of acute liver injury (liver cell necrosis, transaminases) and fibrosis (Sirus red positive area and hydroxyproline content) compared to vehicle treated mice. Ameliorated fibrosis by 44AANA47-CCL5 was associated with a decreased expression of fibrosis related genes, decreased α-smoth muscle antigen (αSMA) and a reduction of infiltrating immune cells. In the acute model, 44AANA47-CCL5 treated mice displayed a reduced immune cell infiltration and mRNA levels of TNF, IL-1 and CCL3 compared to vehicle treated mice. In vitro, conditioned medium of T-cell enriched splenocytes of 44AANA47-CCL5 treated mice inhibited the chemotaxis and proliferation of HSC. Conclusions The results provide evidence that inhibition of oligomerization and glycosaminoglycan binding of the chemokine CCL5 is a new therapeutic strategy for the treatment of acute and chronic liver injuries and represents an alternative to chemokine receptor antagonism.