IL-1β and TNFα Differentially Influence NF-κB Activity and FasL-Induced Apoptosis in Primary Murine Hepatocytes During LPS-Induced Inflammation

Macrophage-derived cytokines largely influence the behavior of hepatocytes during an inflammatory response. We previously reported that both TNFα and IL-1β, which are released by macrophages upon LPS stimulation, affect Fas ligand (FasL)-induced apoptotic signaling. Whereas TNFα preincubation leads to elevated levels of caspase-3 activity and cell death, pretreatment with IL-1β induces increased caspase-3 activity but keeps cells alive. We now report that IL-1β and TNFα differentially influence NF-κB activity resulting in a differential upregulation of target genes, which may contribute to the distinct effects on cell viability. A reduced NF-κB activation model was established to further investigate the molecular mechanisms which determine the distinct cell fate decisions after IL-1β and TNFα stimulation. To study this aspect in a more physiological setting, we used supernatants from LPS-stimulated bone marrow-derived macrophages (BMDMs). The treatment of hepatocytes with the BMDM supernatant, which contains both IL-1β and TNFα, sensitized to FasL-induced caspase-3 activation and cell death. However, when TNFα action was blocked by neutralizing antibodies, cell viability after stimulation with the BMDM supernatant and FasL increased as compared to single FasL stimulation. This indicates the important role of TNFα in the sensitization of apoptosis in hepatocytes. These results give first insights into the complex interplay between macrophages and hepatocytes which may influence life/death decisions of hepatocytes during an inflammatory reaction of the liver in response to a bacterial infection

Improving metabolic parameters of antipsychotic child treatment (IMPACT) study: rationale, design, and methods

BACKGROUND: Youth with serious mental illness may experience improved psychiatric stability with second generation antipsychotic (SGA) medication treatment, but unfortunately may also experience unhealthy weight gain adverse events. Research on weight loss strategies for youth who require ongoing antipsychotic treatment is quite limited. The purpose of this paper is to present the design, methods, and rationale of the Improving Metabolic Parameters in Antipsychotic Child Treatment (IMPACT) study, a federally funded, randomized trial comparing two pharmacologic strategies against a control condition to manage SGA-related weight gain. METHODS: The design and methodology considerations of the IMPACT trial are described and embedded in a description of health risks associated with antipsychotic-related weight gain and the limitations of currently available research. RESULTS: The IMPACT study is a 4-site, six month, randomized, open-label, clinical trial of overweight/obese youth ages 8–19 years with pediatric schizophrenia-spectrum and bipolar-spectrum disorders, psychotic or non-psychotic major depressive disorder, or irritability associated with autistic disorder. Youth who have experienced clinically significant weight gain during antipsychotic treatment in the past 3 years are randomized to either (1) switch antipsychotic plus healthy lifestyle education (HLE); (2) add metformin plus HLE; or (3) HLE with no medication change. The primary aim is to compare weight change (body mass index z-scores) for each pharmacologic intervention with the control condition. Key secondary assessments include percentage body fat, insulin resistance, lipid profile, psychiatric symptom stability (monitored independently by the pharmacotherapist and a blinded evaluator), and all-cause and specific cause discontinuation. This study is ongoing, and the targeted sample size is 132 youth. CONCLUSION: Antipsychotic-related weight gain is an important public health issue for youth requiring ongoing antipsychotic treatment to maintain psychiatric stability. The IMPACT study provides a model for pediatric research on adverse event management using state-of-the art methods. The results of this study will provide needed data on risks and benefits of two pharmacologic interventions that are already being used in pediatric clinical settings but that have not yet been compared directly in randomized trials. TRIAL REGISTRATION: Clinical Trials.gov NCT0080623

Tumor necrosis factor sensitizes primary murine hepatocytes to Fas/CD95-induced apoptosis in a Bim- and Bid-dependent manner

Fas/CD95 is a critical mediator of cell death in many chronic and acute liver diseases and induces apoptosis in primary hepatocytes in vitro. In contrast, the proinflammatory cytokine tumor necrosis factor α (TNFα) fails to provoke cell death in isolated hepatocytes but has been implicated in hepatocyte apoptosis during liver diseases associated with chronic inflammation. Here we report that TNFα sensitizes primary murine hepatocytes cultured on collagen to Fas ligand (FasL)-induced apoptosis. This synergism is time-dependent and is specifically mediated by TNFα. Fas itself is essential for the sensitization, but neither Fas up-regulation nor endogenous FasL is responsible for this effect. Although FasL is shown to induce Bid-independent apoptosis in hepatocytes cultured on collagen, the sensitizing effect of TNFα is clearly dependent on Bid. Moreover, both c-Jun N-terminal kinase activation and Bim, another B cell lymphoma 2 homology domain 3 (BH3)-only protein, are crucial mediators of TNFα-induced apoptosis sensitization. Bim and Bid activate the mitochondrial amplification loop and induce cytochrome c release, a hallmark of type II apoptosis. The mechanism of TNFα-induced sensitization is supported by a mathematical model that correctly reproduces the biological findings. Finally, our results are physiologically relevant because TNFα also induces sensitivity to agonistic anti-Fas-induced liver damage. CONCLUSION: Our data suggest that TNFα can cooperate with FasL to induce hepatocyte apoptosis by activating the BH3-only proteins Bim and Bid

ON/OFF and beyond--a boolean model of apoptosis.

Apoptosis is regulated by several signaling pathways which are extensively linked by crosstalks. Boolean or logical modeling has become a promising approach to capture the qualitative behavior of such complex networks. Here we built a large-scale literature-based Boolean model of the central intrinsic and extrinsic apoptosis pathways as well as pathways connected with them. The model responds to several external stimuli such as Fas ligand, TNF-alpha, UV-B irradiation, interleukin-1beta and insulin. Timescales and multi-value node logic were used and turned out to be indispensable to reproduce the behavior of the apoptotic network. The coherence of the model was experimentally validated. Thereby an UV-B dose-effect is shown for the first time in mouse hepatocytes. Analysis of the model revealed a tight regulation emerging from high connectivity and spanning crosstalks and a particular importance of feedback loops. An unexpected feedback from Smac release to RIP could further increase complex II formation. The introduced Boolean model provides a comprehensive and coherent description of the apoptosis network behavior. It gives new insights into the complex interplay of pro- and antiapoptotic factors and can be easily expanded to other signaling pathways

IL-1β and TNFα differentially influence NF-κB activity and FasL-induced apoptosis in primary murine hepatocytes during LPS-induced inflammation

Macrophage-derived cytokines largely influence the behavior of hepatocytes during an inflammatory response. We previously reported that both TNFα and IL-1β, which are released by macrophages upon LPS stimulation, affect Fas ligand (FasL)-induced apoptotic signaling. Whereas TNFα preincubation leads to elevated levels of caspase-3 activity and cell death, pretreatment with IL-1β induces increased caspase-3 activity but keeps cells alive. We now report that IL-1β and TNFα differentially influence NF-κB activity resulting in a differential upregulation of target genes, which may contribute to the distinct effects on cell viability. A reduced NF-κB activation model was established to further investigate the molecular mechanisms which determine the distinct cell fate decisions after IL-1β and TNFα stimulation. To study this aspect in a more physiological setting, we used supernatants from LPS-stimulated bone marrow-derived macrophages (BMDMs). The treatment of hepatocytes with the BMDM supernatant, which contains both IL-1β and TNFα, sensitized to FasL-induced caspase-3 activation and cell death. However, when TNFα action was blocked by neutralizing antibodies, cell viability after stimulation with the BMDM supernatant and FasL increased as compared to single FasL stimulation. This indicates the important role of TNFα in the sensitization of apoptosis in hepatocytes. These results give first insights into the complex interplay between macrophages and hepatocytes which may influence life/death decisions of hepatocytes during an inflammatory reaction of the liver in response to a bacterial infection

TNFα sensitizes hepatocytes to FasL-induced apoptosis by NFκB-mediated Fas upregulation

Although it is well established that TNFα contributes to hepatitis, liver failure and associated hepatocarcinogenesis via the regulation of inflammation, its pro-apoptotic role in the liver has remained enigmatic. On its own, TNFα is unable to trigger apoptosis. However, when combined with the transcriptional inhibitor GaLN, it can cause hepatocyte apoptosis and liver failure in mice. Moreover, along with others, we have shown that TNFα is capable of sensitizing cells to FasL- or drug-induced cell death via c-Jun N-terminal kinase (JNK) activation and phosphorylation/activation of the BH3-only protein Bim. In this context, TNFα could exacerbate hepatocyte cell death during simultaneous inflammatory and T-cell-mediated immune responses in the liver. Here we show that TNFα sensitizes primary hepatocytes, established hepatocyte cell lines and mouse embryo fibroblasts to FasL-induced apoptosis by the transcriptional induction and higher surface expression of Fas via the NFκB pathway. Genetic deletion, diminished expression or dominant-negative inhibition of the NFκB subunit p65 resulted in lower Fas expression and inhibited TNFα-induced Fas upregulation and sensitization to FasL-induced cell death. By hydrodynamic injection of p65 shRNA into the tail vein of mice, we confirm that Fas upregulation by TNFα is also NFκB-mediated in the liver. In conclusion, TNFα sensitization of FasL-induced apoptosis in the liver proceeds via two parallel signaling pathways, activation of JNK and Bim phosphorylation and NFκB-mediated Fas upregulation.publishe

Simulation of caspase-8 and -3 activities, cytochrome c release and levels of pJNK, Bid, Bim, Bcl-2 and XIAP after treating wt hepatocytes with IL-1β, FasL or a combination of both.

<p>Simulation results for wt cells after stimulation with IL-1β (A, D), FasL (B, E) or both (C, F). IL-1β stimulus is given at time point 0, FasL is added after 12 h. Abbreviations: C8p18, active caspase-8; C3*, active caspase-3; pJNK, phosphorylated JNK.</p

Interleukin-1β Enhances FasL-Induced Caspase-3/-7 Activity without Increasing Apoptosis in Primary Mouse Hepatocytes

<div><p>Sustained inflammation may increase the susceptibility of hepatocytes to apoptotic cell death and therefore exacerbate liver damage. Here we report that the pro-inflammatory cytokine IL-1β sensitizes primary murine hepatocytes to Fas ligand (FasL)-induced caspase-3/-7 activity. This process was dependent on JNK1/2 and the BH3-only proteins Bim and Bid. Mathematical modeling revealed that incubation of hepatocytes with IL-1β depleted the anti-apoptotic Bcl-2 protein pool and thus shifted hepatocytes to mitochondrial type II apoptosis following Fas activation. As a consequence, IL-1β and FasL treatment enhanced cytochrome c release. Surprisingly, despite increased caspase-3/-7 activation, FasL-induced cell death was reduced by IL-1β pre-treatment. This protective effect was independent of JNK1/2, Bim or Bid. Furthermore, elevated caspase-3/-7 activity upon IL-1β and FasL treatment did not result in enhanced PARP cleavage. The protective effect of IL-1β was seen after 3 h of pre-incubation, indicating an anti-apoptotic transcriptional response. Indeed, NF-κB DNA binding was increased in response to IL-1β plus FasL and gene-expression profiling of NF-κB regulated genes revealed a transcriptional and translational upregulation of the caspase-8 inhibitor A20. A mathematical model was developed to explain the contradictious occurrence of both increased caspase-3/-7 activity and elevated cell viability by including a heterogeneous distribution of Bcl-2 proteins and variations in Fas signaling resulting in different subpopulations of hepatocytes.</p></div

Structure of the IL-1β/FasL model.

<p>Inputs of the model are IL-1β and FasL depicted in black and model output is activated caspase-3 (C3*). The model is based on ordinary differential equation (ODE) and mass action kinetics. The integrated NF-κB model from Lipniacki et al. (Lipnicaki et al., 2004) is summarized as a gray box for clarity; the detailed scheme is depicted in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0115603#pone.0115603.s008" target="_blank">S8 Fig</a>. Degradation of species is indicated by boxes with dashed border.</p

NF-κB activation is decreased after combinatorial stimulation with TNFα + FasL, but persists after IL-1β + FasL stimulation.

<p>Nuclear extracts of primary hepatocytes were prepared after treatment with IL-1β (20 ng/ml), TNFα (25 ng/ml), and FasL (50 ng/ml) for the indicated times. Additionally, cells were pre-treated for 30 min with the pan-caspase inhibitor Q-VD-OPh (20 µM) followed by IL-1β or IL-1β + FasL stimulation. Control cells remained untreated (lane 1). Equal nuclear protein amounts were analyzed for NF-κB DNA binding activity in an EMSA.•○ NF-κB-DNA complexes, ▪ non-specific binding to the probe, ◊ unbound oligonucleotide. The result of the EMSA was reproduced and one representative EMSA is shown.</p