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.

Deficiency of formyl peptide receptor 1 and 2 is associated with increased inflammation and enhanced liver injury after LPS-stimulation.

INTRODUCTION: Formyl peptide-receptor 1 and 2 (FPR1 and FPR2) in mice were identified as receptors with contrary affinity for the PAMP fMLF. Formyl-methionyl-leucyl-phenylalanine is either part of the bacterial membrane and is secreted by the mitochondria of eukaryotic ceslls during apoptosis. Furthermore FPR1 and 2 are described as highly relevant factors for the chemotaxis of immune cells. Their role during the acute liver injury has not been investigated yet. MATERIALS AND METHODS: Constitutive knockout mice for FPR1 (mFPR1-/-), FPR2 (mFPR2-/-) and wild type (WT) mice were challenged with LPS i.p. for 3 h and 6 h. Liver and serum were sampled for further analysis. RESULTS: Liver transaminases were elevated in all mice 3 h and 6 h post LPS stimulation. Gene expression analysis displayed a reduced expression of the pro-inflammatory cytokines IL-6 and CXCL1 after 3 h in the mFPR1-/- compared to wild type and mFPR2-/- mice. After 6 h, IL-6, TNF-α and CXCL1 were significantly higher in mice lacking mFPR1 or 2. Consistent to these findings the numbers of CD11b+ and Ly6G+ immune cells were altered in the livers. The analysis of TLR2 and TLR4 revealed time and genotype specific changes in theirs gene expression. Additionally, the liver in mFPR1- and mFPR2-deficient mice seem to be more susceptible to apoptosis by showing a significant higher number of TUNEL+-cells in the liver than WT-mice and displayed less Ki67-positive nuclei in the liver. CONCLUSION: The results suggest a prominent role of FPRs in the regulation of the hepatic inflammatory response after LPS induced liver injury. Deletion of mFPR1 or mFPR2 leads to deregulation of the inflammatory response compared to WT mice, associated with more severe liver injury represented by higher levels of transaminases, apoptotic cells and a reduced regenerative capacity.

Lack of hepatic c-Met and gp130 expression is associated with an impaired antibacterial response and higher lethality after bile duct ligation.

The prognosis of liver failure is often determined by infectious and cholestatic complications. As HGF/c-Met and interleukin (IL)-6/gp130 control hepatic cytoprotective pathways, we here investigated their cooperative role during the onset of cholestatic liver injury. Conditional hepatocyte-specific ((Δhepa)) c-Met, gp130 and c-Met/gp130 knockout mice (Cre-loxP system) were subjected to bile duct ligation (BDL) and lipopolysaccharide (LPS) stimulation. gp130(Δhepa) and c-Met/gp130(Δhepa) mice displayed increased lethality associated with severe bacteraemia early after BDL, whereas c-Met(Δhepa) and wild-type mice showed normal survival. Analysis of the innate immune response and the regulation of hepatic antibacterial pathways showed that the LPS-triggered hepatocellular response via the Toll-like receptor-4 pathway was regulated differentially by HGF/c-Met and IL-6/gp130. Activation of p38MAPK, c-Jun N-terminal kinase and signalling transducer and activator of transcription-3 was impaired in gp130(Δ) and c-Met(Δhepa) livers. In addition, the acute-phase response (APR) was reduced in c-Met(Δhepa) livers, whereas gp130(Δhepa) displayed a completely abolished APR. In contrast, TNF-α-dependent NF-κB activation was enhanced in gp130(Δhepa) and c-Met(Δhepa) mice and it was associated with a higher rate of apoptosis and inflammation. Moreover, expression of the neutrophil produced and secreted cathelin-related antimicrobial peptide and of genes related to the inflammasome complex correlated with the strength of the bacterial infection and with TNF-α expression. In conclusion, Gp130 and c-Met are involved in the hepatic antibacterial and innate immune response, control the APR and thus prevent sepsis and liver injury during cholestatic conditions.

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.

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.

c-Met confers protection against chronic liver tissue damage and fibrosis progression after bile duct ligation in mice.

BACKGROUND & AIMS: The hepatocyte growth factor (HGF)/mesenchymal-epithelial transition factor (c-Met) system is an essential inducer of hepatocyte growth and proliferation. Although a fundamental role for the HGF receptor c-Met has been shown in acute liver regeneration, its cell-specific role in hepatocytes during chronic liver injury and fibrosis progression has not been determined. METHODS: Hepatocyte-specific c-Met knockout mice (c-Met(Delta hepa)) using the Cre-loxP system were studied in a bile duct ligation (BDL) model. Microarray analyses were performed to define HGF/c-Met-dependent gene expression. RESULTS: Two strategies for c-Met deletion in hepatocytes to generate hepatocyte-specific c-Met knockout mice were tested. Early deletion during embryonic development was lethal, whereas post-natal Cre expression was successful, leading to the generation of viable c-Met(Delta hepa) mice. BDL in these mice resulted in extensive necrosis and lower proliferation rates of hepatocytes. Gene array analysis of c-Met(Delta hepa) mice revealed a significant reduction of anti-apoptotic genes in c-Met-deleted hepatocytes. These findings could be tested functionally because c-Met(Delta hepa) mice showed a stronger apoptotic response after BDL and Jo-2 stimulation. The phenotype was associated with increased expression of proinflammatory cytokines (tumor necrosis factor-alpha and interleukin-6) and an enhanced recruitment of neutrophils. Activation of these mechanisms triggered a stronger profibrogenic response as evidenced by increased transforming growth factor-beta(1), alpha-smooth muscle actin, collagen-1alpha messenger RNA expression, and enhanced collagen-fiber staining in c-Met(Delta hepa) mice. CONCLUSIONS: Our results show that deletion of c-Met in hepatocytes leads to more liver cell damage and fibrosis in a chronic cholestatic liver injury model because c-Met triggers survival signals important for hepatocyte recovery.