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.

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.

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.

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.

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

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 CCl(4)-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 TNFalpha 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 CCl(4) 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.

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.

A functional variation in CHI3L1 is associated with severity of liver fibrosis and YKL-40 serum levels in chronic hepatitis C infection.

BACKGROUND/AIMS: YKL-40 is a chitinase-like protein involved in matrix remodelling and a non-invasive fibrosis marker. We assessed whether a functional promoter polymorphism in CHI3L1, encoding YKL-40, is associated with HCV-induced liver fibrosis and influences YKL-40 serum concentrations. METHODS: The CHI3L1 -131G-->C promoter polymorphism was genotyped in two cohorts of HCV infected patients (n=440) by 5'-endonuclease assays. Histological fibrosis scores and YKL-40 serum levels (ELISA) were associated with CHI3L1 -131G-->C by quantitative and qualitative genetic analyses and corrected by multivariate analysis. RESULTS: CHI3L1 -131G-->C genotype was strongly associated with the stage of liver fibrosis in the screening (n=265, P=0.001) and validation cohort (n=175, P=0.009). Homozygous carriers of the G allele were protected from severe fibrosis (F3/F4). This association was confirmed after correction for age and gender. Functionally, the G allele was associated with reduced serum levels of YKL-40 in HCV infected patients (P=0.002). CONCLUSIONS: The CHI3L1 promoter polymorphism -131G-->C determines YKL-40 serum levels and is associated with the severity of HCV-induced liver fibrosis. These results suggest a functional role of YKL-40 in liver fibrogenesis and should be taken into account when using YKL-40 as a non-invasive fibrosis marker.

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.

The fractalkine receptor CX3CR1 is involved in liver fibrosis due to chronic hepatitis C infection.

BACKGROUND/AIMS: The chemokine receptor CX3CR1 and its specific ligand fractalkine (CX3CL1) are known to modulate inflammatory and fibroproliferative diseases. Here we investigate the role of CX3CR1/fractalkine in HCV-induced liver fibrosis. METHODS: A genotype analysis of CX3CR1 variants was performed in 211 HCV-infected patients. Hepatic expression of CX3CR1 was studied in HCV-infected livers and isolated liver cell populations by RT-PCR and immunohistochemistry. The effects of fractalkine on mRNA expression of profibrogenic genes were determined in isolated hepatic stellate cells (HSC) and CX3CR1 genotypes were related to intrahepatic TIMP-1 mRNA levels. RESULTS: The intrahepatic mRNA expression of CX3CR1 correlates with the stage of HCV-induced liver fibrosis (P=0.03). The CX3CR1 coding variant V249I is associated with advanced liver fibrosis, independent of the T280M variant (P=0.009). CX3CR1 is present on primary HSC and fractalkine leads to a suppression of tissue inhibitor of metalloproteinase (TIMP)-1 mRNA in HSC (P=0.03). Furthermore, CX3CR1 genotypes are associated with TIMP-1 mRNA expression in HCV-infected liver (P=0.03). CONCLUSIONS: The results identify the fractalkine receptor CX3CR1 as susceptibility a gene for hepatic fibrosis in HCV infection. The modulation of TIMP-1 expression by fractalkine and CX3CR1 genotypes provides functional support for the observed genotype-phenotype association.