Marie-Luise Berres

BRAF-V600E expression in precursor versus differentiated dendritic cells defines clinically distinct LCH risk groups

The Rockefeller University Press

Antagonism of the chemokine Ccl5 ameliorates experimental liver fibrosis in mice

30.00 J. Exp. Med. 2014 Vol. 211 No. 4 669-683 www.jem.org/cgi/doi/10.1084/jem.20130977 669 Langerhans cell histiocytosis (LCH) is characterized by inflammatory lesions that include pathological langerin+ DCs. LCH has pleotropic clinical presentations ranging from single lesions cured by curettage to potentially fatal multisystem disease. The first descriptions of LCH, including Hand-Schüller-Christian disease and Letter-Siwe disease, were based on anatomical location and extent of the lesions (Arceci, 1999). The diagnosis of high-risk LCH, defined by involvement of “risk organs” which include BM, liver, and spleen, conferred mortality rates >20%, where patients with disease limited to non-risk organs (low-risk LCH) had nearly 100% survival, CORRESPONDENCE Carl Allen: ceallen@txch.org OR Miriam Merad: miriam.merad@mssm.edu

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

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.

Inhibition of hepatic fibrogenesis by matrix metalloproteinase-9 mutants in mice

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

Tissue inhibitor of metalloproteinases‐1 (TIMP‐1) plays a crucial role in the pathogenesis of hepatic fibrosis and thus may represent an important therapeutic target in the design of anti‐fibrotic strategies for chronic liver disease. We present an innovative therapy based on the assignment of inactivated enzymes acting as scavengers for TIMP‐1. Hepatic fibrosis was induced in BALB/c mice by repetitive intraperitoneal CCl4 injection. The animals were treated with proteolytic inactive matrix metalloproteinase‐9 mutants (E402Q, H401A, E402H/H411E) using adenovirus‐mediated gene transfer. Application of these MMP‐9 mutants inhibited fibrogenesis, which was indicated by decreasing portal and periportal accumulation of collagen. Total hydroxyproline of liver tissue, the morphometric stage of fibrosis as well as mRNA expression of marker proteins for hepatic fibrosis in livers of E402Qand H401A‐treated mice were significantly reduced. MMP‐9 mutants suppressed transdifferentiation of hepatic stellate cells to the myofibroblast like phenotype in vitro and in vivo. Moreover, adenoviral application of the mutants MMP‐9‐H401A and ‐E402Q led to increased apoptosis of activated hepatic stellate cells, thought to be the main promoters of hepatic fibrosis. Application of MMP‐9 mutants as TIMP‐1 scavengers may provide a new therapeutic strategy for hepatic fibrosis.—Roderfeld, M., Weiskirchen, R., Wagner, S., Berres, M.‐L., Henkel, C., Grötzinger, J., Gressner, A. M., Matern, S., Roeb, E. Inhibition of hepatic fibrogenesis by matrix metalloproteinase‐9 mutants in mice. FASEB J. 20, 444–454 (2006)

Chemokine Cxcl9 attenuates liver fibrosis‐associated angiogenesis in mice

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.)

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

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)

Longitudinal monocyte human leukocyte antigen-DR expression is a prognostic marker in critically ill patients with decompensated liver cirrhosis.

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.

Progress in understanding the pathogenesis of Langerhans cell histiocytosis: back to Histiocytosis X?

Background: Critical illness in cirrhotic patients is associated with a poor prognosis and increased susceptibility to infections. Monocyte HLA‐DR expression is decreased in cirrhotic patients, but its prognostic value has not been investigated prospectively.

Serum chemokine receptor CXCR3 ligands are associated with progression, organ dysfunction and complications of chronic liver diseases

Langerhans cell histiocytosis (LCH), the most common histiocytic disorder, is characterized by the accumulation of CD1A+/CD207+ mononuclear phagocytes within granulomatous lesions that can affect nearly all organ systems. Historically, LCH has been presumed to arise from transformed or pathologically activated epidermal dendritic cells called Langerhans cells. However, new evidence supports a model in which LCH occurs as a consequence of a misguided differentiation programme of myeloid dendritic cell precursors. Genetic, molecular and functional data implicate activation of the ERK signalling pathway at critical stages in myeloid differentiation as an essential and universal driver of LCH pathology. Based on these findings, we propose that LCH should be re‐defined as an inflammatory myeloid neoplasia. Increased understanding of LCH pathogenesis will provide opportunities to optimize and personalize therapy through improved risk‐stratification, targeted therapy and assessment of therapy response based on specific molecular features and origin of the pathological myeloid cells.