Konrad L. Streetz

Fatality in mice due to oversaturation of cellular microRNA/short hairpin RNA pathways

RNA interference (RNAi) is a universal and evolutionarily conserved phenomenon of post-transcriptional gene silencing by means of sequence-specific mRNA degradation, triggered by small double-stranded RNAs. Because this mechanism can be efficiently induced in vivo by expressing target-complementary short hairpin RNA (shRNA) from non-viral and viral vectors, RNAi is attractive for functional genomics and human therapeutics. Here we systematically investigate the long-term effects of sustained high-level shRNA expression in livers of adult mice. Robust shRNA expression in all the hepatocytes after intravenous infusion was achieved with an optimized shRNA delivery vector based on duplex-DNA-containing adeno-associated virus type 8 (AAV8). An evaluation of 49 distinct AAV/shRNA vectors, unique in length and sequence and directed against six targets, showed that 36 resulted in dose-dependent liver injury, with 23 ultimately causing death. Morbidity was associated with the downregulation of liver-derived microRNAs (miRNAs), indicating possible competition of the latter with shRNAs for limiting cellular factors required for the processing of various small RNAs. In vitro and in vivo shRNA transfection studies implied that one such factor, shared by the shRNA/miRNA pathways and readily saturated, is the nuclear karyopherin exportin-5. Our findings have fundamental consequences for future RNAi-based strategies in animals and humans, because controlling intracellular shRNA expression levels will be imperative. However, the risk of oversaturating endogenous small RNA pathways can be minimized by optimizing shRNA dose and sequence, as exemplified here by our report of persistent and therapeutic RNAi against human hepatitis B virus in vivo.

The Mitochondrial Permeability Transition Is Required for Tumor Necrosis Factor Alpha-Mediated Apoptosis and Cytochrome c Release

ABSTRACT This study assesses the controversial role of the mitochondrial permeability transition (MPT) in apoptosis. In primary rat hepatocytes expressing an IκB superrepressor, tumor necrosis factor alpha (TNFα) induced apoptosis as shown by nuclear morphology, DNA ladder formation, and caspase 3 activation. Confocal microscopy showed that TNFα induced onset of the MPT and mitochondrial depolarization beginning 9 h after TNFα treatment. Initially, depolarization and the MPT occurred in only a subset of mitochondria; however, by 12 h after TNFα treatment, virtually all mitochondria were affected. Cyclosporin A (CsA), an inhibitor of the MPT, blocked TNFα-mediated apoptosis and cytochrome c release. Caspase 3 activation, cytochrome c release, and apoptotic nuclear morphological changes were induced after onset of the MPT and were prevented by CsA. Depolarization and onset of the MPT were blocked in hepatocytes expressing ΔFADD, a dominant negative mutant of Fas-associated protein with death domain (FADD), or crmA, a natural serpin inhibitor of caspases. In contrast, Asp-Glu-Val-Asp-cho, an inhibitor of caspase 3, did not block depolarization or onset of the MPT induced by TNFα, although it inhibited cell death completely. In conclusion, the MPT is an essential component in the signaling pathway for TNFα-induced apoptosis in hepatocytes which is required for both cytochrome c release and cell death and functions downstream of FADD and crmA but upstream of caspase 3.

Interleukin 6 and liver regeneration

The multifunctional cytokine interleukin 6 (IL-6) was cloned in 1986. Since then, our understanding of its role in the regulatory functions in the immune system has grown steadily. IL-6 is a typical pleiotropic cytokine that acts on various cells. It is commonly produced at local tissue sites and is released in almost all situations of homeostatic perturbation, which include endotoxaemia, trauma, and acute infection. In addition, circulating IL-6, together with tumour necrosis factor α (TNF-α) and interleukin 1 (IL-1), is required for induction of the acute phase response which comprises fever, corticosteroid release, and hepatic production of acute phase proteins which are mostly protease inhibitors. Overall, induction of the acute phase response by IL-6 has been regarded as part of an attempt to maintain homeostasis. Apart from its role in inflammation, IL-6 induces differentiation and development of B cells, T cells, myeloid cells, megakaryocytes, osteoclasts, neural cells, and hepatocytes. IL-6 acts as a growth factor for renal cell carcinoma and Kaposis sarcoma, and promotes the growth of haematopoietic stem cells. Apart from the cytokine itself, the IL-6 family comprises IL-11, ciliary neurotrophic factor (CNTF), cardiotropin (CT-1), oncostatin M (OSM), leukaemia inhibitory factor (LIF), and the novel neurotrophin 1/B cell stimulating factor 3 (NNT-1/BSF-3), which all share the common signal transducer gp130 as part of their receptors. Except for CNTFR, the IL-6-type cytokine signalling receptors are type I membrane proteins (extracellular N terminus, one transmembrane domain). This receptor family is defined by the presence of at least one cytokine binding molecule (CBM), a set of four conserved tyrosine residues, and a tryptophan-serine-X-tryptophan-serine (W-S-X-S-W) motif, located outside the transmembrane domain. Signal transduction after ligand binding is elicited by homodimerisation of gp130 in the case of IL-6 and IL-11, by heterodimerisation of gp130 and LIFR in the case of LIF, CNTF and CT-1, …

Interleukin-6/glycoprotein 130-dependent pathways are protective during liver regeneration.

After tissue loss the liver has the unique capacity to restore its mass by hepatocyte proliferation. Interleukin-6 (IL6)-deficient mice show a lack in DNA synthesis after partial hepatectomy (PH). To define better the role of IL6 and its family members for liver regeneration after PH, we used conditional knockout mice for glycoprotein 130 (gp130), the common signal transducer of all IL6 family members. We show that gp130-dependent pathways control Stat3 activation after PH. By using gene array analysis, we demonstrate that c-jun, NF-κB, c-myc, and tumor necrosis factor receptor expression is gp130-dependent. However, in gp130-deleted mice only minor effects on cell cycle and on the maximum of DNA synthesis after PH were found compared with controls. As in conditional gp130 animals, the acute phase response was completely abolished, we considered that other means are essential to define the role of gp130-dependent pathways for liver regeneration. LPS stimulation in gp130-deleted and also IL6 −/− animals after PH leads to a significant reduction in survival and DNA synthesis, which was associated with decreased Bcl-xL expression and higher apoptosis in the liver. These results indicate that the phenotype concerning the reduction in DNA synthesis might be linked to the degree of infection after PH. Thus our results suggest that the role of gp130-dependent signaling is not a direct influence on cell cycle progression after partial hepatectomy but is to activate protective pathways important to enable hepatocyte proliferation.

Experimental liver fibrosis research: update on animal models, legal issues and translational aspects.

Liver fibrosis is defined as excessive extracellular matrix deposition and is based on complex interactions between matrix-producing hepatic stellate cells and an abundance of liver-resident and infiltrating cells. Investigation of these processes requires in vitro and in vivo experimental work in animals. However, the use of animals in translational research will be increasingly challenged, at least in countries of the European Union, because of the adoption of new animal welfare rules in 2013. These rules will create an urgent need for optimized standard operating procedures regarding animal experimentation and improved international communication in the liver fibrosis community. This review gives an update on current animal models, techniques and underlying pathomechanisms with the aim of fostering a critical discussion of the limitations and potential of up-to-date animal experimentation. We discuss potential complications in experimental liver fibrosis and provide examples of how the findings of studies in which these models are used can be translated to human disease and therapy. In this review, we want to motivate the international community to design more standardized animal models which might help to address the legally requested replacement, refinement and reduction of animals in fibrosis research.

Concanavalin A—induced liver cell damage: Activation of intracellular pathways triggered by tumor necrosis factor in mice

BACKGROUND & AIMS Concanavalin A (con A) induces tumor necrosis factor (TNF)-dependent hepatocyte apoptosis resembling immune-mediated fulminant hepatic failure in humans. Intracellular pathways originating at the TNF receptor are either linked to apoptosis, nuclear factor (NF)-kappaB translocation, or Jun kinase (JNK) activation. The aim of this study was to study TNF-dependent pathways after con A injection in vivo. METHODS Con A, con A plus anti-TNF, and control buffer were injected into BALB/c mice. Immunofluorescence, Western blot, Northern blot, gel shift, Erk, and JNK activity and DNA fragmentation experiments were performed at different time points after injection. RESULTS DNA fragmentation in hepatocytes was increased 4-24 hours after con A injection. JNK was activated maximally (>20-fold) directly after con A injection, whereas binding and nuclear translocation of NF-kappaB was maximal after 4 hours. All pathways were blocked by anti-TNF. JNK activation was specific because related ERK 1 + 2 were not activated after con A. High nuclear expression of c-Jun was already evident 1 hour after con A injection; however, in contrast to JNK, anti-TNF treatment did not block c-Jun nuclear expression and DNA binding. CONCLUSIONS In the con A model, activation of TNF-dependent pathways is associated with apoptosis of hepatocytes. Their modulation in vivo may have implications to develop new therapeutic strategies to prevent apoptosis.

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.

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

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.

Interleukin-6-induced Tethering of STAT3 to the LAP/C/EBPβ Promoter Suggests a New Mechanism of Transcriptional Regulation by STAT3

LAP/C/EBPβ is a member of the C/EBP family of transcription factors and contributes to the regulation of the acute phase response in hepatocytes. Here we show that IL-6 controlsLAP/C/EBPβ gene transcription and identify an IL-6 responsive element in the LAP/C/EBPβ promoter, which contains no STAT3 DNA binding motif. However, luciferase reporter gene assays showed that STAT3 activation through the gp130 signal transducer molecule is involved in mediating IL-6-dependent LAP/C/EBPβ transcription. Southwestern analysis indicated that IL-6 induces binding of a 68-kDa protein to the recently characterized CRE-like elements in the LAP/C/EBPβ promoter. Transfection experiments using promoter constructs with mutated CRE-like elements revealed that these sites confer IL-6 responsiveness. Further analysis using STAT1/STAT3 chimeras identified specific domains of the protein that are required for the IL-6-dependent increase inLAP/C/EBPβ gene transcription. Overexpression of the amino-terminal domain of STAT3 blocked the IL-6-mediated response, suggesting that the STAT3 amino terminus has an important function in IL-6-mediated transcription of the LAP/C/EBPβ gene. These data lead to a model of how tethering STAT3 to a DNA-bound complex contributes to IL-6-dependent LAP/C/EBPβ gene transcription. Our analysis describes a new mechanism by which STAT3 controls gene transcription and which has direct implication for the acute phase response in liver cells.

CXC chemokine ligand 4 (Cxcl4) is a platelet‐derived mediator of experimental liver fibrosis

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.)