Daniel Heinrichs

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.

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 CCL3 Promotes Experimental Liver Fibrosis in Mice

Liver fibrosis is associated with infiltrating immune cells and activation of hepatic stellate cells. We here aimed to investigate the effects of the CC chemokine CCL3, also known as macrophage inflammatory protein-1α, in two different fibrosis models. To this end, we treated mice either with carbon tetrachloride or with a methionine- and choline-deficient diet to induce fibrosis in CCL3 deficient and wild-type mice. The results show that the protein expression of CCL3 is increased in wild-type mice after chronic liver injury. Deletion of CCL3 exhibited reduced liver fibrosis compared to their wild-type counterparts. We could validate these results by treating the two mouse groups with either carbon tetrachloride or by feeding a methionine- and choline-deficient diet. In these models, lack of CCL3 is functionally associated with reduced stellate cell activation and liver immune cell infiltration. In vitro, we show that CCL3 leads to increased proliferation and migration of hepatic stellate cells. In conclusion, our results define the chemokine CCL3 as a mediator of experimental liver fibrosis. Thus, therapeutic modulation of CCL3 might be a promising target for chronic liver diseases.

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.—Heinrichs, D., Berres, M.‐L., Coeuru, M., Knauel, M., Nellen, A., Fischer, P., Philippeit, C., Bucala, R., Trautwein, C., Wasmuth, H. E., Bernhagen, J., Protective role of macrophage migration inhibitory factor in nonalcoholic steatohepatitis. FASEB J. 28, 5136–5147 (2014). www.fasebj.org

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.

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 (CCl4) 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-β, 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 CCl4 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.

The Anti-Microbial Peptide LL-37/CRAMP Is Elevated in Patients with Liver Diseases and Acts as a Protective Factor during Mouse Liver Injury

Background: Antimicrobial peptides (AMP) are an important defense mechanism of the innate immune system and can modulate the course of various diseases. However, their significance during liver pathogenesis is currently not well defined. Methods: Patients with liver diseases were analyzed for LL-37/CRAMP, human beta-defensin-2 (hBD2), and complement 5a (C5a) serum levels. Mice deficient in CRAMP (Cathelicidin-related Antimicrobial Peptide), the mouse homolog for human LL-37, were fed with a methionine- and choline-deficient diet (MCD) and underwent bile-duct ligation (BDL). Results: First, serum samples from patients with chronic liver diseases were investigated. Therefore, significantly enhanced levels for LL-37, hBD2, and complement C5a were detected, all of which comprise antimicrobial properties. Next, CRAMP-knockout (CRAMP-KO) mice were investigated, to better define a functional role of LL-37/CRAMP in animal models of liver diseases. MCD feeding and bile-duct ligation of CRAMP-KO mice resulted in an enhanced degree of liver injury during the early treatment phase. MCD feeding in CRAMP-KO mice led to stronger intrahepatic fat accumulation and significantly enhanced matrix remodeling, whereas BDL caused more extensive liver necrosis. At the late 28 days time point, MCD-fed CRAMP-KO mice displayed a higher intrahepatic fat load. Long-term changes in bile-duct-ligated mice included higher collagen content as a sign of enhanced fibrosis progression if CRAMP was absent. Conclusion: The study shows a clear correlation of antimicrobial peptide serum levels in patients with chronic liver diseases. Furthermore, we were able to demonstrate protective functions of LL-37/CRAMP in two independent mouse models of chronic liver injury.

Characterization of adipose tissue macrophages and adipose-derived stem cells in critical wounds

Background Subcutaneous adipose tissue is a rich source of adipose tissue macrophages and adipose-derived stem cells which both play a key role in wound repair. While macrophages can be divided into the classically-activated M1 and the alternatively-activated M2 phenotype, ASCs are characterized by the expression of specific stem cell markers. Methods In the present study, we have investigated the expression of common macrophage polarization and stem cell markers in acutely inflamed adipose tissue. Subcutaneous adipose tissue adjacent to acutely inflamed wounds of 20 patients and 20 healthy subjects were harvested and underwent qPCR and flow cytometry analysis. Results Expression levels of the M1-specific markers CD80, iNOS, and IL-1b were significantly elevated in inflammatory adipose tissue when compared to healthy adipose tissue, whereas the M2-specific markers CD163 and TGF-β were decreased. By flow cytometry, a significant shift of adipose tissue macrophage populations towards the M1 phenotype was confirmed. Furthermore, a decrease in the mesenchymal stem cell markers CD29, CD34, and CD105 was observed whereas CD73 and CD90 remained unchanged. Discussion This is the first report describing the predominance of M1 adipose tissue macrophages and the reduction of stem cell marker expression in acutely inflamed, non-healing wounds.

Contents Vol. 91, 2015

Founded as ‘Archiv für Verdauungskrankheiten’ 1895 by I. Boas Continued as ‘Gastroenterologia’ 1939–1967 Former Editors: P. Morawitz (1934–1936), R. Staehelin (1937–1943), A. Hurst (1940–1945), W. Löffler (1943–1961), T.C. Hunt (1947–1967), N. Henning (1953–1962), B. Ihre (1953–1967), H. Bartelheimer (1963–1967), M. Demole (1963–1971), H. Kapp (1968–1970), R. Lambert (1972–1978), W. Creutzfeldt (1979–1992), R. Arnold (1993–2003), C. Beglinger (2004–2011), B. Göke (2004–2014)