Sebastian Huss

Pharmacological inhibition of the chemokine CCL2 (MCP-1) diminishes liver macrophage infiltration and steatohepatitis in chronic hepatic injury

Objective Monocyte chemoattractant protein-1 (MCP-1, CCL2), the primary ligand for chemokine receptor C–C chemokine receptor 2 (CCR2), is increased in livers of patients with non-alcoholic steatohepatitis (NASH) and murine models of steatohepatitis and fibrosis. It was recently shown that monocyte/macrophage infiltration into the liver upon injury is critically regulated by the CCL2/CCR2 axis and is functionally important for perpetuating hepatic inflammation and fibrogenesis. The structured L-enantiomeric RNA oligonucleotide mNOX-E36 (a so-called Spiegelmer) potently binds and inhibits murine MCP-1. Pharmacological inhibition of MCP-1 with mNOX-E36 was investigated in two murine models of chronic liver diseases. Methods Pharmacological inhibition of MCP-1 by thrice-weekly mNOX-E36 subcutaneously was tested in murine models of acute or chronic carbon tetrachloride (CCl4)- and methionine–choline-deficient (MCD) diet-induced chronic hepatic injury in vivo. Results Antagonising MCP-1 by mNOX-E36 efficiently inhibited murine monocyte chemotaxis in vitro as well as migration of Gr1+ (Ly6C+) blood monocytes into the liver upon acute toxic injury in vivo. In murine models of CCl4- and MCD diet-induced hepatic injury, the infiltration of macrophages into the liver was significantly decreased in anti-MCP-1-treated mice as found by fluorescence-activated cell sorting (FACS) analysis and immunohistochemistry. In line with lower levels of intrahepatic macrophages, proinflammatory cytokines (tumour necrosis factor α, interferon γ and interleukin 6) were significantly reduced in liver tissue. Overall fibrosis progression over 6 (CCl4) or 8 weeks (MCD diet) was not significantly altered by anti-MCP-1 treatment. However, upon MCD diet challenge a lower level of fatty liver degeneration (histology score, Oil red O staining, hepatic triglyceride content, lipogenesis genes) was detected in mNOX-E36-treated animals. mNOX-E36 also ameliorated hepatic steatosis upon therapeutic administration. Conclusions These results demonstrate the successful pharmacological inhibition of hepatic monocyte/macrophage infiltration by blocking MCP-1 during chronic liver damage in two in vivo models. The associated ameliorated steatosis development suggests that inhibition of MCP-1 is an interesting novel approach for pharmacological treatment in liver inflammation and steatohepatitis.

Definition of a fluorescence in-situ hybridization score identifies high- and low-level FGFR1 amplification types in squamous cell lung cancer

We recently reported fibroblast growth factor receptor-type 1 (FGFR1) amplification to be associated with therapeutically tractable FGFR1 dependency in squamous cell lung cancer. This makes FGFR1 a novel target for directed therapy in these tumors. To reproducibly identify patients for clinical studies, we developed a standardized reading and evaluation strategy for FGFR1 fluorescence in-situ hybridization (FISH) and propose evaluation criteria, describe different patterns of low- and high-level amplifications and report on the prevalence of FGFR1 amplifications in pulmonary carcinomas. A total of 420 lung cancer patients including 307 squamous carcinomas, 100 adenocarcinomas of the lung and 13 carcinomas of other types were analyzed for FGFR1 amplification using a dual color FISH. We found heterogeneous and different patterns of gene copy numbers. FGFR1 amplifications were observed in 20% of pulmonary squamous carcinomas but not in adenocarcinomas. High-level amplification (as defined by an FGFR1/centromer 8 (CEN8) ratio ≥2.0, or average number of FGFR1 signals per tumor cell nucleus ≥6, or the percentage of tumor cells containing ≥15 FGFR1 signals or large clusters ≥10%) was detected at a frequency of 16% and low-level amplification (as defined by ≥5 FGFR1 signals in ≥50% of tumor cells) at a frequency of 4%. We conclude that FGFR1 amplification is one of the most frequent therapeutically tractable genetic lesions in pulmonary carcinomas. Standardized reporting of FGFR1 amplification in squamous carcinomas of the lung will become increasingly important to correlate therapeutic responses with FGFR1 inhibitors in clinical studies. Thus, our reading and evaluation strategy might serve as a basis for identifying patients for ongoing and upcoming clinical trials.

Hepatic macrophage migration and differentiation critical for liver fibrosis is mediated by the chemokine receptor C‐C motif chemokine receptor 8 in mice

Chemokines critically control the infiltration of immune cells upon liver injury, thereby promoting hepatic inflammation and fibrosis. The chemokine receptor CCR8 can affect trafficking of monocytes/macrophages, monocyte‐derived dendritic cells (DCs) and T‐helper cell (Th) subsets, but its role in liver diseases is currently unknown. To investigate the functional role of CCR8 in liver diseases, ccr8−/− and wild‐type (WT) mice were subjected to chronic experimental injury models of carbon tetrachloride (CCl4) administration and surgical bile duct ligation (BDL). CCR8 was strongly up‐regulated in the injured liver. Ccr8−/− mice displayed attenuated liver damage (e.g., ALT, histology, and TUNEL) compared to WT mice and were also protected from liver fibrosis in two independent injury models. Flow cytometry revealed reduced infiltrates of liver macrophages, neutrophils and natural killer cells, whereas hepatic CD4+ T cells increased. The main CCR8‐expressing cells in the liver were hepatic macrophages, and CCR8 was functionally necessary for CCL1‐directed migration of inflammatory but not for nonclassical monocytes into the liver. Moreover, the phenotype of liver macrophages from injured ccr8−/− animals was altered with increased expression of DC markers and enhanced expression of T‐cell‐attracting chemokine macrophage inflammatory protein 1‐alpha (MIP‐1α/CCL3). Correspondingly, hepatic CD4+ T cells showed increased Th1 polarization and reduced Th2 cells in CCR8‐deficient animals. Liver fibrosis progression, but also subsequent T‐cell alterations, could be restored by adoptively transferring CCR8‐expressing monocytes/macrophages into ccr8−/− mice during experimental injury. Conclusions: CCR8 critically mediates hepatic macrophage recruitment upon injury, which subsequently shapes the inflammatory response in the injured liver, affecting macrophage/DC and Th differentiation. CCR8 deficiency protects the liver against injury, ameliorating initial inflammatory responses and hepatic fibrogenesis. Inhibition of CCR8 or its ligand, CCL1, might represent a successful therapeutic target to limit liver inflammation and fibrosis progression. (Hepatology 2012)

Chemokine Receptor CCR6-Dependent Accumulation of γδ T Cells in Injured Liver Restricts Hepatic Inflammation and Fibrosis

Chronic liver injury promotes hepatic inflammation, representing a prerequisite for organ fibrosis. We hypothesized a contribution of chemokine receptor CCR6 and its ligand, CCL20, which may regulate migration of T‐helper (Th)17, regulatory, and gamma‐delta (γδ) T cells. CCR6 and CCL20 expression was intrahepatically up‐regulated in patients with chronic liver diseases (n = 50), compared to control liver (n = 5). Immunohistochemistry revealed the periportal accumulation of CCR6+ mononuclear cells and CCL20 induction by hepatic parenchymal cells in liver disease patients. Similarly, in murine livers, CCR6 was expressed by macrophages, CD4 and γδ T‐cells, and up‐regulated in fibrosis, whereas primary hepatocytes induced CCL20 upon experimental injury. In two murine models of chronic liver injury (CCl4 and methionine‐choline‐deficient diet), Ccr6−/− mice developed more severe fibrosis with strongly enhanced hepatic immune cell infiltration, compared to wild‐type (WT) mice. Although CCR6 did not affect hepatic Th‐cell subtype composition, CCR6 was explicitly required by the subset of interleukin (IL)‐17‐ and IL‐22‐expressing γδ T cells for accumulation in injured liver. The adoptive transfer of WT γδ, but not CD4 T cells, into Ccr6−/− mice reduced hepatic inflammation and fibrosis in chronic injury to WT level. The anti‐inflammatory function of hepatic γδ T cells was independent of IL‐17, as evidenced by transfer of Il‐17−/− cells. Instead, hepatic γδ T cells colocalized with hepatic stellate cells (HSCs) in vivo and promoted apoptosis of primary murine HSCs in a cell‐cell contact‐dependent manner, involving Fas‐ligand (CD95L). Consistent with γδ T‐cell‐induced HSC apoptosis, activated myofibroblasts were more frequent in fibrotic livers of Ccr6−/− than in WT mice. Conclusion: γδ T cells are recruited to the liver by CCR6 upon chronic injury and protect the liver from excessive inflammation and fibrosis by inhibiting HSCs. (Hepatology 2014;59:630–642)

β‐Catenin (CTNNB1) mutations and clinicopathological features of mesenteric desmoid‐type fibromatosis

Aims and methods:  Desmoid‐type fibromatosis (desmoid) is a fibroblastic tumour that shows locally aggressive growth. Mesenteric desmoid is a rare lesion that shares morphological and biological features with fibromatoses occurring in the abdominal wall or in extraabdominal sites, but differs in terms of gross appearance and clinical presentation. We report on a series of 56 cases of mesenteric desmoids from our consultation files and compare them with cases of non‐mesenteric desmoids and retroperitoneal fibrosis.

Role of cannabinoid receptors in alcoholic hepatic injury: steatosis and fibrogenesis are increased in CB2 receptor-deficient mice and decreased in CB1 receptor knockouts

Background: Alcohol is a common cause of hepatic liver injury with steatosis and fibrosis. Cannabinoid receptors (CB) modulate steatosis, inflammation and fibrogenesis. To investigate the differences between CB1 and CB2 in the hepatic response to chronic alcohol intake, we examined CB knockout mice (CB1−/−, CB2−/−).

HSC-specific inhibition of Rho-kinase reduces portal pressure in cirrhotic rats without major systemic effects

BACKGROUND & AIMS Rho-kinase activation mediates cell contraction and increases intrahepatic resistance and consequently portal pressure in liver cirrhosis. Systemic Rho-kinase inhibition decreases portal pressure in cirrhosis, but also arterial pressure. Thus, liver-specific Rho-kinase inhibition is needed. The delivery of Rho-kinase inhibitor to activated hepatic stellate cells reduces fibrosis. It might also relax these contractile cells and therewith decrease intrahepatic resistance. We tested this hypothesis by performing acute experiments in cirrhotic rats. METHODS Cirrhosis models were CCl(4)-intoxication and bile duct ligation. Three hours after injection of the Rho-kinase inhibitor (Y26732) coupled with a carrier (mannose-6-phosphate modified human serum albumin), which targets activated hepatic stellate cells, hemodynamics were analyzed by the colored microsphere technique and direct pressure measurements. The delivery site and effect of Rho-kinase inhibitor were investigated by immunohistochemical stainings, as well as Western blot. Experiments with Rho-kinase inhibitor coupled with unmodified human serum albumin served as untargeted control. RESULTS In both models of cirrhosis, the carrier coupled Rho-kinase inhibitor lowered the portal pressure and decreased the hepatic-portal resistance. Immunohistochemical desmin-staining showed the carrier in hepatic stellate cells. The targeted therapy decreased the expression of the phosphorylated substrate of Rho-kinase (moesin) and abolished myosin light chains phosphorylation in fibrotic septae (collagen-staining). The targeted Rho-kinase inhibitor showed no major extrahepatic effects. By contrast, the untargeted Rho-kinase inhibitor elicited severe systemic hypotension. CONCLUSIONS Activated hepatic stellate cells are crucially involved in portal hypertension in cirrhosis. Targeting of Rho-kinase in hepatic stellate cells not only decreased fibrosis, as previously shown, but also lowers portal pressure acutely without major systemic effects as demonstrated in this study.

Angiotensin‐II type 1 receptor‐mediated Janus kinase 2 activation induces liver fibrosis

Activation of the renin angiotensin system resulting in stimulation of angiotensin‐II (AngII) type I receptor (AT1R) is an important factor in the development of liver fibrosis. Here, we investigated the role of Janus kinase 2 (JAK2) as a newly described intracellular effector of AT1R in mediating liver fibrosis. Fibrotic liver samples from rodents and humans were compared to respective controls. Transcription, protein expression, activation, and localization of JAK2 and downstream effectors were analyzed by real‐time polymerase chain reaction, western blotting, immunohistochemistry, and confocal microscopy. Experimental fibrosis was induced by bile duct ligation (BDL), CCl4 intoxication, thioacetamide intoxication or continuous AngII infusion. JAK2 was inhibited by AG490. In vitro experiments were performed with primary rodent hepatic stellate cells (HSCs), Kupffer cells (KCs), and hepatocytes as well as primary human and human‐derived LX2 cells. JAK2 expression and activity were increased in experimental rodent and human liver fibrosis, specifically in myofibroblastic HSCs. AT1R stimulation in wild‐type animals led to activation of HSCs and fibrosis in vivo through phosphorylation of JAK2 and subsequent RhoA/Rho‐kinase activation. These effects were prevented in AT1R−/− mice. Pharmacological inhibition of JAK2 attenuated liver fibrosis in rodent fibrosis models. In vitro, JAK2 and downstream effectors showed increased expression and activation in activated HSCs, when compared to quiescent HSCs, KCs, and hepatocytes isolated from rodents. In primary human and LX2 cells, AG490 blocked AngII‐induced profibrotic gene expression. Overexpression of JAK2 led to increased profibrotic gene expression in LX2 cells, which was blocked by AG490. Conclusion: Our study substantiates the important cell‐intrinsic role of JAK2 in HSCs for development of liver fibrosis. Inhibition of JAK2 might therefore offer a promising therapy for liver fibrosis. (Hepatology 2014;60:334–348)

Activating PDGFRA mutations in inflammatory fibroid polyps occur in exons 12, 14 and 18 and are associated with tumour localization

Huss S, Wardelmann E, Goltz D, Binot E, Hartmann W, Merkelbach‐Bruse S, Büttner R & Schildhaus H‐U (2012) Histopathology 61, 59–68

Correlation of Intraprostatic Tumor Extent with 68Ga-PSMA Distribution in Patients with Prostate Cancer

We evaluated the diagnostic value and accuracy of prostate-specific membrane antigen (PSMA) PET for the intraprostatic delineation of prostate cancer before prostatectomy. Methods: We identified 6 patients with biopsy-proven high-risk prostate cancer who were referred for 68Ga-PSMA PET/CT before radical prostatectomy to rule out metastasis. After prostatectomy, a histologic map of the prostate was reconstructed. The histologic extent and Gleason score of each segment of the prostate were compared with 68Ga-PSMA PET images resliced to the histologic axis. Sensitivity, specificity, positive and negative predictive value, and positive and negative likelihood ratios were calculated. The SUV of each segment was measured, and median values were compared. Results: Of the 132 segments, 112 were eligible for analysis. The correlation of histologic results with 68Ga-PSMA PET images showed a specificity and sensitivity of 92%. The positive and negative likelihood ratio and the positive and negative predictive value for detection of prostate cancer on 68Ga-PSMA PET were 11.5, 0.09, 96%, and 85%, respectively. The median SUVmax of true-positive prostate segments was significantly higher than that of true-negative segments (11.0 ± 7.8 vs. 2.7 ± 0.9, P < 0.001), and a cutoff of 4 revealed a sensitivity and specificity of 86.5% and an accuracy of 87.5%. Conclusion: These preliminary results show that the intraprostatic localization and extent of prostate cancer may be estimated by 68Ga-PSMA PET. This imaging method may be helpful for identifying target lesions before prostate biopsy and may support decision making before focal or radical therapy.