Robust RNAi enhancement via human Argonaute-2 overexpression from plasmids, viral vectors and cell lines.

As the only mammalian Argonaute protein capable of directly cleaving mRNAs in a small RNA-guided manner, Argonaute-2 (Ago2) is a keyplayer in RNA interference (RNAi) silencing via small interfering (si) or short hairpin (sh) RNAs. It is also a rate-limiting factor whose saturation by si/shRNAs limits RNAi efficiency and causes numerous adverse side effects. Here, we report a set of versatile tools and widely applicable strategies for transient or stable Ago2 co-expression, which overcome these concerns. Specifically, we engineered plasmids and viral vectors to co-encode a codon-optimized human Ago2 cDNA along with custom shRNAs. Furthermore, we stably integrated this Ago2 cDNA into a panel of standard human cell lines via plasmid transfection or lentiviral transduction. Using various endo- or exogenous targets, we demonstrate the potential of all three strategies to boost mRNA silencing efficiencies in cell culture by up to 10-fold, and to facilitate combinatorial knockdowns. Importantly, these robust improvements were reflected by augmented RNAi phenotypes and accompanied by reduced off-targeting effects. We moreover show that Ago2/shRNA-co-encoding vectors can enhance and prolong transgene silencing in livers of adult mice, while concurrently alleviating hepatotoxicity. Our customizable reagents and avenues should broadly improve future in vitro and in vivo RNAi experiments in mammalian systems.

Proapoptotic effects of the chemokine, CXCL 10 are mediated by the noncognate receptor TLR4 in hepatocytes.

UNLABELLED: Aberrant expression of the chemokine CXC chemokine ligand (CXCL)10 has been linked to the severity of hepatitis C virus (HCV)-induced liver injury, but the underlying molecular mechanisms remain unclear. In this study, we describe a yet-unknown proapoptotic effect of CXCL10 in hepatocytes, which is not mediated through its cognate chemokine receptor, but the lipopolysaccharide receptor Toll-like receptor 4 (TLR4). To this end, we investigated the link of CXCL10 expression with apoptosis in HCV-infected patients and in murine liver injury models. Mice were treated with CXCL10 or neutralizing antibody to systematically analyze effects on hepatocellular apoptosis in vivo. Direct proapoptotic functions of CXCL10 on different liver cell types were evaluated in detail in vitro. The results showed that CXCL10 expression was positively correlated with liver cell apoptosis in humans and mice. Neutralization of CXCL10 ameliorated concanavalin A-induced tissue injury in vivo, which was strongly associated with reduced liver cell apoptosis. In vitro, CXCL10 mediated the apoptosis of hepatocytes involving TLR4, but not CXC chemokine receptor 3 signaling. Specifically, CXCL10 induced long-term protein kinase B and Jun N-terminal kinase activation, leading to hepatocyte apoptosis by caspase-8, caspase-3, and p21-activated kinase 2 cleavage. Accordingly, systemic application of CXCL10 led to TLR4-induced liver cell apoptosis in vivo. CONCLUSION: The results identify CXCL10 and its noncognate receptor, TLR4, as a proapoptotic signaling cascade during liver injury. Antagonism of the CXCL10/TLR4 pathway might be a therapeutic option in liver diseases associated with increased apoptosis.

Albumin is recycled from the primary urine by tubular transcytosis.

Under physiologic conditions, significant amounts of plasma protein pass the renal filter and are reabsorbed by proximal tubular cells, but it is not clear whether the endocytosed protein, particularly albumin, is degraded in lysosomes or returned to the circulatory system intact. To resolve this question, a transgenic mouse with podocyte-specific expression of doxycycline-inducible tagged murine albumin was developed. To assess potential glomerular backfiltration, two types of albumin with different charges were expressed. On administration of doxycycline, podocytes expressed either of the two types of transgenic albumin, which were secreted into the primary filtrate and reabsorbed by proximal tubular cells, resulting in serum accumulation. Renal transplantation experiments confirmed that extrarenal transcription of transgenic albumin was unlikely to account for these results. Genetic deletion of the neonatal Fc receptor (FcRn), which rescues albumin and IgG from lysosomal degradation, abolished transcytosis of both types of transgenic albumin and IgG in proximal tubular cells. In summary, we provide evidence of a transcytosis within the kidney tubular system that protects albumin and IgG from lysosomal degradation, allowing these proteins to be recycled intact.

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 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.

Glycoprotein 130-dependent pathways in host hepatocytes are important for liver repopulation in mice.

Hepatocyte transplantation (HT) is still restricted by the limited amount of transplantable cells. Therefore, a better understanding of the mechanisms involved in cellular engraftment, proliferation, and in vivo selection is important. Here we aimed to evaluate the role of the interleukin 6 (IL-6)/glycoprotein 130 (gp130) system for liver repopulation. Mice carrying a conditional hepatocyte-specific deletion of the common IL-6 signal transducer gp130 (gp130(Deltahepa)) were used for HT. First, we compared bone marrow transplantation (BMT), partial hepatectomy (PH), and retrorsine treatment of recipient mice to optimize the in vivo selection of transplanted hepatocytes. BMT combined with PH was sufficient to induce a 30-fold increase in the number of transplanted donor hepatocytes, whereas additional retrorsine pretreatment led to an up to 40-fold increase. Next, the influence of gp130 signaling in hepatocytes on cell selection was evaluated. Wild-type (WT) hepatocytes repopulated WT recipients at the same rate as gp130(Deltahepa) cells. In contrast, liver repopulation by transplanted cells was enhanced in gp130(Deltahepa) recipient mice. This was associated with higher proliferation of donor hepatocytes and enhanced apoptosis in gp130(Deltahepa) recipient livers. Additionally, the acute phase response was strongly induced after HT in WT recipients but blunted in gp130(Deltahepa) recipients. As a result, significantly more liver remodeling, evidenced by stronger hepatic stellate cell activation and collagen accumulation, was found in gp130(Deltahepa) mice after HT. In conclusion, the HT model established here can be efficiently applied to investigate cell-specific mechanisms in liver repopulation. Moreover, we have shown that gp130-dependent pathways in host hepatocytes are important for controlling liver repopulation.

Hepatic tissue environment in NEMO-deficient mice critically regulates positive selection of donor cells after hepatocyte transplantation.

BACKGROUND: Hepatocyte transplantation (HT) is a promising alternative treatment strategy for end-stage liver diseases compared with orthotopic liver transplantation. A limitation for this approach is the low engraftment of donor cells. The deletion of the I-kappa B kinase-regulatory subunit IKKγ/NEMO in hepatocytes prevents nuclear factor (NF)-kB activation and triggers spontaneous liver apoptosis, chronic hepatitis and the development of liver fibrosis and hepatocellular carcinoma. We hypothesized that NEMOΔhepa mice may therefore serve as an experimental model to study HT. METHODS: Pre-conditioned NEMOΔhepa mice were transplanted with donor-hepatocytes from wildtype (WT) and mice deficient for the pro-apoptotic mediator Caspase-8 (Casp8Δhepa). RESULTS: Transplantation of isolated WT-hepatocytes into pre-conditioned NEMOΔhepa mice resulted in a 6-7 fold increase of donor cells 12 weeks after HT, while WT-recipients showed no liver repopulation. The use of apoptosis-resistant Casp8Δhepa-derived donor cells further enhanced the selection 3-fold after 12-weeks and up to 10-fold increase after 52 weeks compared with WT donors. While analysis of NEMOΔhepa mice revealed strong liver injury, HT-recipient NEMOΔhepa mice showed improved liver morphology and decrease in serum transaminases. Concomitant with these findings, the histological examination elicited an improved liver tissue architecture associated with significantly lower levels of apoptosis, decreased proliferation and a lesser amount of liver fibrogenesis. Altogether, our data clearly support the therapeutic benefit of the HT procedure into NEMOΔhepa mice. CONCLUSION: This study demonstrates the feasibility of the NEMOΔhepa mouse as an in vivo tool to study liver repopulation after HT. The improvement of the characteristic phenotype of chronic liver injury in NEMOΔhepa mice after HT suggests the therapeutic potential of HT in liver diseases with a chronic inflammatory phenotype and opens a new door for the applicability of this technique to combat liver disease in the human clinic.