Tom Luedde

Senescence surveillance of pre-malignant hepatocytes limits liver cancer development.

Upon the aberrant activation of oncogenes, normal cells can enter the cellular senescence program, a state of stable cell-cycle arrest, which represents an important barrier against tumour development in vivo. Senescent cells communicate with their environment by secreting various cytokines and growth factors, and it was reported that this ‘secretory phenotype’ can have pro- as well as anti-tumorigenic effects. Here we show that oncogene-induced senescence occurs in otherwise normal murine hepatocytes in vivo. Pre-malignant senescent hepatocytes secrete chemo- and cytokines and are subject to immune-mediated clearance (designated as ‘senescence surveillance’), which depends on an intact CD4+ T-cell-mediated adaptive immune response. Impaired immune surveillance of pre-malignant senescent hepatocytes results in the development of murine hepatocellular carcinomas (HCCs), thus showing that senescence surveillance is important for tumour suppression in vivo. In accordance with these observations, ras-specific Th1 lymphocytes could be detected in mice, in which oncogene-induced senescence had been triggered by hepatic expression of NrasG12V. We also found that CD4+ T cells require monocytes/macrophages to execute the clearance of senescent hepatocytes. Our study indicates that senescence surveillance represents an important extrinsic component of the senescence anti-tumour barrier, and illustrates how the cellular senescence program is involved in tumour immune surveillance by mounting specific immune responses against antigens expressed in pre-malignant senescent cells.

Micro‐RNA profiling reveals a role for miR‐29 in human and murine liver fibrosis

Liver fibrosis is orchestrated by a complex network of signaling pathways regulating the deposition of extracellular matrix proteins during fibrogenesis. MicroRNAs (miRNAs) represent a family of small noncoding RNAs controlling translation and transcription of many genes. Recently, miRNAs have been suggested to crucially modulate cellular processes in the liver such as hepatocarcinogenesis. However, their role in liver fibrosis is not well understood. We systematically analyzed the regulation of miRNAs in a mouse model of carbon tetrachloride–induced hepatic fibrogenesis (CCl4) by gene array analysis, which revealed a panel of miRNA that were specifically regulated in livers of mice undergoing hepatic fibrosis. Within those, all three members of the miR‐29‐family were significantly down‐regulated in livers of CCl4‐treated mice as well as in mice that underwent bile duct ligation. Specific regulation of miR‐29 members in murine fibrosis models correlated with lower expression of miR‐29 in livers from patients with advanced liver fibrosis. Moreover, patients with advanced liver cirrhosis showed significantly lower levels of miR‐29a in their serum when compared with healthy controls or patients with early fibrosis. On a cellular level, down‐regulation of miR‐29 in murine hepatic stellate cells (HSCs) was mediated by transforming growth factor beta (TGF‐β) as well as inflammatory signals, namely, lipopolysaccharide (LPS) and nuclear factor kappa B (NF‐κB). Furthermore, overexpression of miR‐29b in murine HSC resulted in down‐regulation of collagen expression. Conclusion: Our data indicate that miR‐29 mediates the regulation of liver fibrosis and is part of a signaling nexus involving TGF‐β‐ and NF‐κB–dependent down‐regulation of miR‐29 family members in HSC with subsequent up‐regulation of extracellular matrix genes. Thus they may represent targets for novel therapeutic strategies against hepatic fibrogenesis and also might evolve as biomarkers in the diagnosis of liver fibrosis. (HEPATOLOGY 2011.)

Hepatic Recruitment of the Inflammatory Gr1+ Monocyte Subset Upon Liver Injury Promotes Hepatic Fibrosis

In addition to liver‐resident Kupffer cells, infiltrating immune cells have recently been linked to the development of liver fibrosis. Blood monocytes are circulating precursors of tissue macrophages and can be divided into two functionally distinct subpopulations in mice: Gr1hi (Ly6Chi) and Gr1lo (Ly6Clo) monocytes. The role of these monocyte subsets in hepatic fibrosis and the mechanisms of their differential recruitment into the injured liver are unknown. We therefore characterized subpopulations of infiltrating monocytes in acute and chronic carbon tetrachloride (CCl4)‐induced liver injury in mice using flow cytometry and immunohistochemistry. Inflammatory Gr1hi but not Gr1lo monocytes are massively recruited into the liver upon toxic injury constituting an up to 10‐fold increase in CD11b+F4/80+ intrahepatic macrophages. Comparing wild‐type with C‐C chemokine receptor (CCR2)‐deficient and CCR2/CCR6–deficient mice revealed that CCR2 critically controls intrahepatic Gr1hi monocyte accumulation by mediating their egress from bone marrow. During chronic liver damage, intrahepatic CD11b+F4/80+Gr1+ monocyte‐derived cells differentiate preferentially into inducible nitric oxide synthase–producing macrophages exerting proinflammatory and profibrogenic actions, such as promoting hepatic stellate cell (HSC) activation, T helper 1–T cell differentiation and transforming growth factor β (TGF‐β) release. Impaired monocyte subset recruitment in Ccr2−/− and Ccr2−/−Ccr6−/− mice results in reduced HSC activation and diminished liver fibrosis. Moreover, adoptively transferred Gr1hi monocytes traffic into the injured liver and promote fibrosis progression in wild‐type and Ccr2−/−Ccr6−/− mice, which are otherwise protected from hepatic fibrosis. Intrahepatic CD11b+F4/80+Gr1+ monocyte‐derived macrophages purified from CCl4‐treated animals, but not naïve bone marrow monocytes or control lymphocytes, directly activate HSCs in a TGF‐β–dependent manner in vitro. Conclusion: Inflammatory Gr1+ monocytes, recruited into the injured liver via CCR2‐dependent bone marrow egress, promote the progression of liver fibrosis. Thus, they may represent an interesting novel target for antifibrotic strategies. (HEPATOLOGY 2009;50:261–274.)

NF-κB in the liver—linking injury, fibrosis and hepatocellular carcinoma

Hepatic cirrhosis and hepatocellular carcinoma (HCC) are the most common causes of death in patients with chronic liver disease. Chronic liver injury of virtually any etiology triggers inflammatory and wound-healing responses that in the long run promote the development of hepatic fibrosis and HCC. Here, we review the role of the transcription factor nuclear factor-κB (NF-κB), a master regulator of inflammation and cell death, in the development of hepatocellular injury, liver fibrosis and HCC, with a particular focus on the role of NF-κB in different cellular compartments of the liver. We propose that NF-κB acts as a central link between hepatic injury, fibrosis and HCC, and that it may represent a target for the prevention or treatment of liver fibrosis and HCC. However, NF-κB acts as a two-edged sword and inhibition of NF-κB may not only exert beneficial effects but also negatively impact hepatocyte viability, especially when NF-κB inhibition is pronounced. Finding appropriate targets or identifying drugs that either exert only a moderate effect on NF-κB activity or that can be specifically delivered to nonparenchymal cells will be essential to avoid the increase in liver injury associated with complete NF-κB blockade in hepatocytes.

Tracheobronchial transplantation with a stem-cell-seeded bioartificial nanocomposite: a proof-of-concept study

BACKGROUND Tracheal tumours can be surgically resected but most are an inoperable size at the time of diagnosis; therefore, new therapeutic options are needed. We report the clinical transplantation of the tracheobronchial airway with a stem-cell-seeded bioartificial nanocomposite. METHODS A 36-year-old male patient, previously treated with debulking surgery and radiation therapy, presented with recurrent primary cancer of the distal trachea and main bronchi. After complete tumour resection, the airway was replaced with a tailored bioartificial nanocomposite previously seeded with autologous bone-marrow mononuclear cells via a bioreactor for 36 h. Postoperative granulocyte colony-stimulating factor filgrastim (10 μg/kg) and epoetin beta (40,000 UI) were given over 14 days. We undertook flow cytometry, scanning electron microscopy, confocal microscopy epigenetics, multiplex, miRNA, and gene expression analyses. FINDINGS We noted an extracellular matrix-like coating and proliferating cells including a CD105+ subpopulation in the scaffold after the reseeding and bioreactor process. There were no major complications, and the patient was asymptomatic and tumour free 5 months after transplantation. The bioartificial nanocomposite has patent anastomoses, lined with a vascularised neomucosa, and was partly covered by nearly healthy epithelium. Postoperatively, we detected a mobilisation of peripheral cells displaying increased mesenchymal stromal cell phenotype, and upregulation of epoetin receptors, antiapoptotic genes, and miR-34 and miR-449 biomarkers. These findings, together with increased levels of regenerative-associated plasma factors, strongly suggest stem-cell homing and cell-mediated wound repair, extracellular matrix remodelling, and neovascularisation of the graft. INTERPRETATION Tailor-made bioartificial scaffolds can be used to replace complex airway defects. The bioreactor reseeding process and pharmacological-induced site-specific and graft-specific regeneration and tissue protection are key factors for successful clinical outcome. FUNDING European Commission, Knut and Alice Wallenberg Foundation, Swedish Research Council, StratRegen, Vinnova Foundation, Radiumhemmet, Clinigene EU Network of Excellence, Swedish Cancer Society, Centre for Biosciences (The Live Cell imaging Unit), and UCL Business.

Inflammatory pathways in liver homeostasis and liver injury.

The liver is a unique organ with respect to its anatomical location, allowing continuous blood flow from the gastrointestinal tract through the sinusoids, and its cellular composition, comprising metabolically active hepatocytes, nonhepatocytic parenchymal cells, and various immune cell populations. Cytokines are key mediators within the complex interplay of intrahepatic immune cells and hepatocytes, as they can activate effector functions of immune cells, as well as hepatocytic intracellular signaling pathways controlling cellular homeostasis. Kupffer cells and liver-infiltrating monocyte-derived macrophages are primary sources of cytokines such as tumor-necrosis factor-alpha (TNF-alpha) and interleukin-6. The liver is also enriched in natural killer (NK) and NK T cells, which fulfill functions in pathogen defense, T cell recruitment, and modulation of liver injury. TNF-alpha can activate specific intracellular pathways in hepatocytes that influence cell fate in different manners, e.g., proapoptotic signals via the caspase cascade, but also survival pathways, namely the nuclear factor (NF)-kappaB pathway. NF-kappaB regulates important functions in liver physiology and pathology. Recent experiments with genetically modified mice demonstrated important and partly controversial functions of this pathway, e.g., in cytokine-mediated hepatocyte apoptosis or ischemia–reperfusion injury. The exact dissection of the contribution of recruited and resident immune cells, their soluble cytokine and chemokine mediators, and the intracellular hepatocytic response in liver homeostasis and injury could potentially identify novel targets for the treatment of acute and chronic liver disease, liver fibrosis, or cirrhosis.

A new type of microglia gene targeting shows TAK1 to be pivotal in CNS autoimmune inflammation

Microglia are brain macrophages and, as such, key immune-competent cells that can respond to environmental changes. Understanding the mechanisms of microglia-specific responses during pathologies is hence vital for reducing disease burden. The definition of microglial functions has so far been hampered by the lack of genetic in vivo approaches that allow discrimination of microglia from closely related peripheral macrophage populations in the body. Here we introduce a mouse experimental system that specifically targets microglia to examine the role of a mitogen-associated protein kinase kinase kinase (MAP3K), transforming growth factor (TGF)-β-activated kinase 1 (TAK1), during autoimmune inflammation. Conditional depletion of TAK1 in microglia only, not in neuroectodermal cells, suppressed disease, significantly reduced CNS inflammation and diminished axonal and myelin damage by cell-autonomous inhibition of the NF-κB, JNK and ERK1/2 pathways. Thus, we found TAK1 to be pivotal in CNS autoimmunity, and we present a tool for future investigations of microglial function in the CNS.

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.

Targeted ablation of IKK2 improves skeletal muscle strength, maintains mass, and promotes regeneration

NF-kappaB is a major pleiotropic transcription factor modulating immune, inflammatory, cell survival, and proliferative responses, yet the relevance of NF-kappaB signaling in muscle physiology and disease is less well documented. Here we show that muscle-restricted NF-kappaB inhibition in mice, through targeted deletion of the activating kinase inhibitor of NF-kappaB kinase 2 (IKK2), shifted muscle fiber distribution and improved muscle force. In response to denervation, IKK2 depletion protected against atrophy, maintaining fiber type, size, and strength, increasing protein synthesis, and decreasing protein degradation. IKK2-depleted mice with a muscle-specific transgene expressing a local Igf-1 isoform (mIgf-1) showed enhanced protection against muscle atrophy. In response to muscle damage, IKK2 depletion facilitated skeletal muscle regeneration through enhanced satellite cell activation and reduced fibrosis. Our results establish IKK2/NF-kappaB signaling as an important modulator of muscle homeostasis and suggest a combined role for IKK inhibitors and growth factors in the therapy of muscle diseases.

Cell Death and Cell Death Responses in Liver Disease: Mechanisms and Clinical Relevance

Hepatocellular death is present in almost all types of human liver disease and is used as a sensitive parameter for the detection of acute and chronic liver disease of viral, toxic, metabolic, or autoimmune origin. Clinical data and animal models suggest that hepatocyte death is the key trigger of liver disease progression, manifested by the subsequent development of inflammation, fibrosis, cirrhosis, and hepatocellular carcinoma. Modes of hepatocellular death differ substantially between liver diseases. Different modes of cell death such as apoptosis, necrosis, and necroptosis trigger specific cell death responses and promote progression of liver disease through distinct mechanisms. In this review, we first discuss molecular mechanisms by which different modes of cell death, damage-associated molecular patterns, and specific cell death responses contribute to the development of liver disease. We then review the clinical relevance of cell death, focusing on biomarkers; the contribution of cell death to drug-induced, viral, and fatty liver disease and liver cancer; and evidence for cell death pathways as therapeutic targets.