Mitochondrion 11 (2011) 8388

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Mitochondrion
j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / m i t o

MitoMatters

Toll-like receptor-3-induced mitochondrial dysfunction in cultured human hepatocytes

Siamak Djafarzadeh a, Madhusudanarao Vuda a, Jukka Takala a, Matthias Ochs b,1, Stephan M. Jakob a,
a b

Department of Intensive Care Medicine, Inselspital, Bern University Hospital and University of Bern, 3010 Bern, Switzerland Institute of Anatomy, University of Bern, 3012 Bern, Switzerland

a r t i c l e

i n f o

a b s t r a c t
Several studies have shown the presence of liver mitochondrial dysfunction during sepsis. TLR3 recognizes viral double-stranded RNA and host endogenous cellular mRNA released from damaged cells. TLR3 ligand amplies the systemic hyperinammatory response observed during sepsis and in sepsis RNA escaping from damaged tissues/cells may serve as an endogenous ligand for TLR3 thereby modulating immune responses. This study addressed the hypothesis that TLR3 might regulate mitochondrial function in cultured human hepatocytes. HepG2 cells were exposed to TLR-3 ligand (dsRNA polyinosinepolycytidylic acid; Poly I:C) and mitochondrial respiration was measured. Poly I:C induced a reduction in maximal mitochondrial respiration of human hepatocytes which was prevented partially by preincubation with cyclosporine A (a mitochondrial permeability transition pore-opening inhibitor). Poly-I:C induced activation of NF-B, and the mitochondrial dysfunction was accompanied by caspase-8 but not caspase-3 activation and by no major alterations in cellular or mitochondrial ultrastructure. 2010 Elsevier B.V. and Mitochondria Research Society. All rights reserved.

Article history: Received 4 December 2009 Received in revised form 19 July 2010 Accepted 23 July 2010 Available online 4 August 2010 Keywords: Toll-like receptor Cyclosporine A Mitochondrial permeability transition Mitochondrial respiration Caspase

1. Introduction Severe sepsis and septic shock are major causes of death in intensive care patients (Dombrovskiy et al., 2007). The involvement of TLR-3 activation during experimental polymicrobial septic peritonitis in the absence of an exogenous viral stimulus has been observed and, in vivo, anti-TLR-3 antibody signicantly decreases sepsis-induced mortality in mice (Cavassani et al., 2008). TLR-3 ligand amplies the systemic hyperinammatory response observed during sepsis (Doughty et al., 2006). The data indicated that both the interferon / and NF-B pathways (actived by treatment of mice with TLR-3 ligand) were critical for potentiation of the inammatory response and lethality induced afterward by bacterial infection/septic shock (Doughty et al., 2006). During sepsis, liver dysfunction is common (Vincent et al., 2006) and contributes to the high mortality observed in these patients (Angus et al., 2001; Russell et al., 2000). Although the precise mechanisms by which the liver is affected are unclear (Elbers and
Abbreviations: TLR, toll-like receptor; CsA, cyclosporine A; poly I:C, polyinosine polycytidylic acid; dsRNA, double-stranded RNA; MPT, mitochondrial permeability transition; NF- B, nuclear factor-kappa B; TMPD, N , N , N , N -tetramethyl- p phenylenediamine. Corresponding author. Department of Intensive Care Medicine, Bern University Hospital, Inselspital, CH-3010 Bern, Switzerland. Tel.: +41 31 632 1176; fax: +41 31 632 9644. E-mail address: stephan.jakob@insel.ch (S.M. Jakob). 1 Present address: Institute of Functional and Applied Anatomy, Hannover Medical School, D-30625 Hannover, Germany.

Ince, 2006), there is indication that the failure of mitochondria to effectively couple oxygen consumption with energy production, which has been described in sepsis (Brealey et al., 2002), may contribute to this pathology. Since the liver is a target for various viral infections, such as hepatitis B and C viruses, it seems reasonable to hypothesize that TLR3-mediated signalling contributes to liver mitochondrial dysfunction. The expression of TLR-3, which responds primarily to nucleic acids and is implicated in viral recognition, has been reported in human liver (Nishimura and Naito, 2005) and hepatic cell lineages (Khvalevsky et al., 2007). DsRNA, which is associated with viral infection and is produced by viruses during their replication cycle, and the standard immunostimulant poly I:C (a synthetic analog of dsRNA and a molecular pattern associated with viral infection) induce innate immune responses by two pathways (Kawai and Akira, 2007). In one pathway, extracellular poly I:C (naked poly I:C) is internalized through endocytosis and recognized by TLR-3 (Alexopoulou et al., 2001; De Miranda et al., 2009; Gitlin et al., 2006; Kato et al., 2005). Upon recognition, TLR-3 induces the activation of NF-B to increase production of type I interferons, which signal other cells to increase their antiviral defenses (Yamamoto et al., 2002). In the second pathway (intracellular pathway), transfected complexed poly I:C (complexes between poly I:C and transfection reagents to facilitate cellular internalization) is recognized by intracellular cytoplasmic sensors melanoma differentiation-associated gene 5 (MDA-5) and retinoic acid inducible gene I (RIG-I), which results in the activation of the transcription factors interferon regulatory factor 3 (IRF-3), NF-kB, and activating protein 1 (AP-1) (Honda and Taniguchi, 2006; Gitlin et al., 2006; Kato et al.,

1567-7249/$ see front matter 2010 Elsevier B.V. and Mitochondria Research Society. All rights reserved. doi:10.1016/j.mito.2010.07.010

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2005). Although TLR3 recognizes viral double-stranded RNA, recent studies have suggested that host endogenous cellular mRNA either released from damaged tissue or contained within endocytosed cells may serve as a ligand for TLR3 (Kariko et al., 2004). This may have important physiologic relevance in conditions such as sepsis, because RNA escaping from damaged tissues/cells may serve as an endogenous ligand for TLR3, thereby modulating immune responses. The aim of this study was to evaluate whether TLR-3, stimulated using naked poly I:C, could regulate mitochondrial function in cultured human hepatocytes and to investigate involved molecular mechanisms. 2. Materials and methods 2.1. Materials Naked Poly I:C was purchased from InvivoGen (San Diego, CA, USA). Cyclosporine A, anti-actin antibody and reagents for cellular respiration were obtained from Sigma-Aldrich (Buchs, Switzerland), caspase-3 antibody from Assay Designs (Ann Arbor, MI, USA). 2.2. Cell culture and immunoblot analysis The human hepatoma cell line HepG2 was cultured and immunoblot analysis was performed as described previously (Regueira et al., 2009). For immunoblot analysis of caspase 3, HepG2 cells were incubated for 4 h with Poly I:C (25 g/ml) or cobalt chloride (1 mM and 3 mM; positive control). 2.3. Cellular respiration (high-resolution respirometry) HepG2 cells were incubated with Poly I:C (25 g/ml) for 2, 4 and 6 h (n = 18 each). For studies concerning inhibition of mitochondrial permeability transition (MPT) pore opening, cells were pretreated with cyclosporine A (CsA) at 1 M for 1 h, followed by incubation with Poly I:C (25 g/ml) for 4 h. Cellular respiration was measured as described previously (Regueira et al., 2009). Glutamate/malate, succinate and ascorbate/TMPD [N,N,N,N-tetramethyl-p-phenylendiamine] were used as substrates to test the function of complex I, complex II and complex IV. A representative diagram of measurement of respiration rates in permeabilized HepG2 cells using highresolution respirometry (Oxygraph-2 k, Oroboros Instruments, Innsbruck, Austria) and DatLab 4.2 software (Oroboros Instruments,

Innsbruck, Austria) for data acquisition and analysis is shown in Fig. 1. Each experiment was performed in two chambers in parallel, one as a control and the other with the pretreated cells for paired comparisons of the slopes of the oxygen concentrations of each individual experiment.

2.4. Determination of NF-B (p65) activity The production of NF-B p65 was measured with a NF-B assay kit (Active Motif Carlsbad, CA, USA) according to the manufacturer's instructions. In brief, cell extracts of unstimulated cells, TLR-3-agonist (dsRNA Poly I:C, 25 g/ml, for 30 min)-induced cells and cells pretreated with CsA at 1 M for 1 h, followed by incubation with Poly I:C (25 g/ml) for 30 min, were added to each well coated with consensus-binding site oligonucleotides of NF-B p65. A primary Ab specic for an epitope on the bound and active form of the transcription factor was then added, followed by subsequent incubation with secondary Ab. Addition of the secondary antibody conjugated to horseradish peroxidase provided sensitive colorimetric readout that was quantied by spectrophotometry.

2.5. Determination of caspase-8 activity Cell extracts were prepared from the unstimulated cells and cells incubated with Poly I:C (25 g/ml) for 4 h pretreated with or without CsA at 1 M for 1 h. Caspase-8 activity was determined using a caspase-8 colorimetric assay kit (Clontech Laboratories, Palo Alto, CA, USA) and the plate was read on a microplate reader using 405 nm wavelength light (OD405nm). The cleavage of synthetic caspase8 substrate IETD-pNA was detected spectrophotometrically by the formation of pNA.

2.6. Transmission electron microscopy Transmission electron microscopy was performed as described previously (Regueira et al., 2009). Mitochondrial ultrastructure was assessed with respect to signs of swelling, intactness of cristae, and matrix density (Vetterlein et al., 2003). Accordingly, assessment of cell ultrastructure was performed by evaluating nuclear chromatin architecture, width of perinuclear cisternae and ER as well as cytoplasmic matrix density.

Fig. 1. Representative diagram of measurement of respiration rates in permeabilized HepG2 cells using high-resolution respirometry. Oxygen consumption is expressed as pmol/s/number of cells. Oxygen concentration (blue line) decreases over time as cells use the available oxygen. The oxygen ux (red line) represents the directly calculated oxygen use. Substrates: complex I-dependent respiration (glutamate and malate), complex II-dependent respiration (succinate after inhibition of complex I with rotenone), complex IV-dependent respiration (ascorbate/ TMPD [N,N,N,N-tetramethyl-p-phenylendiamine]) after inhibition of complex III with antimycin A). State 3: active respiration after addition of ADP.

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2.7. Statistics The SPSS 15.0 software package (SPSS Inc., Chicago, IL, USA) was used for statistical analysis. Normal distribution was conrmed by the KolmogorovSmirnov test. The slopes of oxygen concentrations (cellular respiration) of each individual experiment were compared using paired samples t-tests. Unpaired Student's t tests were performed for evaluation of signicance of NF- B (p65) and caspase-8 activities. All data are presented as mean SD, and a p b 0.05 was considered signicant. 3. Results 3.1. Cellular oxygen consumption after TLR-3-agonist stimulation Representative diagram of measurement of respiration rates in permeabilized HepG2 cells at an experimental density of 1106/ml using high-resolution respirometry is shown in Fig. 1. Respiration of cells was measured under routine concentrations as described in Materials and methods. The oxygen ux (red line) represents the directly calculated oxygen use and is expressed as pmol/s/number of cells. Oxygen concentration (blue line) decreases over time as cells use the available oxygen. After 2 h of incubation, Poly I:C (25 g/ml) induced a signicant reduction of complex IV-dependent respiration (11%: 14353 in controls vs. 122 38 pmol/[s million cells] in stimulated cells; p = 0.005) (Fig. 2a). At 4 h of Poly I:C incubation, a signicant reduction in respiration was observed for complex I- (13%: 11729 in controls vs. 10021 pmol/ [s million cells] in stimulated cells; p = 0.008) and complex IVdependent respiration (23%: 18849 in controls vs. 14236 pmol/ [s million cells] in stimulated cells; p b 0.001) (Fig. 2b). At 6 h of Poly I:C incubation, a signicant reduction in cellular respiration was observed only for complex IV-dependent respiration (13%: 16649 in controls vs. 14345 pmol/[s million cells] in stimulated cells; p=0.003) (Fig. 2c). 3.2. Cellular oxygen consumption after inhibition of mitochondrial permeability transition (MPT) pore opening In order to see if the alterations in mitochondrial respiration were linked to the opening of the MPT pores, we tested the effect of cyclosporine A (CsA), an MPT pore inhibitor (Fig. 3). In this series of experiments, at 4 h of Poly I:C incubation, a signicant reduction in respiration was again observed for complex I-dependent (89 16 in controls vs. 83 16 pmol/[s million cells] in stimulated cells; p = 0.028) and complex IV-dependent respiration (110 26 in controls vs. 98 22 pmol/[s million cells] in stimulated cells; p b 0.017). Cells pre-treated with CsA before the addition of Poly I:C (4 h of incubation) exhibited no signicant reduction in complex Idependent respiration in comparison with controls (89 16 in controls vs. 84 15 pmol/[s million cells] in pretreated cells; p = 0.167). CsA did not prevent complex IV-dependent Poly I:Cinduced reduction in cellular respiration (171 26 in controls vs. 97 23 pmol/[s Million cells] in pretreated cells; p = 0.024). CsA alone did not affect complex I-dependent respiration of HepG2 cells but surprisingly induced a signicant reduction in complex IV-dependent respiration. Complex II-dependent respiration was not affected. 3.3. Poly I:C induces activation of NF-B (p65) To assess the effect of Poly I:C on intracellular signaling, we investigated the activation status of transcription factor NF-B (Fig. 4) (n = 4). Treatment with Poly I:C (25 g/ml) for 30 min led to activation of NF-B p65 (0.21 0.02 in controls vs. 0.27 0.02 optical density [O.D.] units at 450 nm (OD 450 nm) in cells treated with Poly I:C; p = 0.006). Preincubation of the cells with CsA at 1 M for 1 h down-regulated Poly I:C (25 g/ml for 30 min)-induced NF-B activation (0.21 0.02 in controls vs. 0.25 0.005 OD 450 nm in cells To determine whether Poly I:C induces activation of caspase-3 apoptosis signaling in HepG2 cells, we examined processing/activation of caspase-3 (Fig. 5b). For these experiments, cells were treated for 4 h with 25 g/ml of Poly I:C. We used an antibody which can recognize both the inactive precursor procaspase-3 (~36 kDa) and the mature active enzymes of 1720 kDa following cleavage by upstream caspases. Relatively little, if any, processing of procaspase-3 to active caspase-3 was detected in untreated cells or in cells treated with Poly I:C. However, treatment of the cells with CoCl2 (3 mM for 4 h;

Fig. 2. HepG2 permeabilized cells' oxygen consumption for complex I, II and IV after 2 (a), 4 (b), and 6 h (c) of incubation with Poly I:C (25 g/ml) (n = 18 each). Data represent mean SD. Statistical signicance between samples using paired t-test is indicated (*p b 0.01 vs. control).

treated with Poly I:C; p = 0.03) although the increase was still statistically signicant. CsA alone did not affect NF-B activity. 3.4. Poly I:C does not induce activation of caspase-3 apoptosis signaling

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Fig. 3. HepG2 permeabilized cells' oxygen consumption for complex I, II and IV, incubated with vehicle for 4 h, cyclosporine A (1 M), Poly I:C (25 g/ml) or preincubated with cyclosporine A for 1 h followed by incubation with Poly I:C (25 g/ml) for an additional 4 h (n = 18). Data represent mean SD. Statistical signicance between samples using paired t-test is indicated (*p b 0.05 vs. control).

positive control) induced a reduction in procaspase-3 protein band intensity with weakly visible cleaved active caspase 3 fragments.

4. Discussion In the current study we used naked dsRNA (poly I:C), which reects a product from viruses during replication, to stimulate TLR-3 (Alexopoulou et al., 2001). Poly(I:C) was added to cells without

3.5. Poly I:C induces activation of caspase-8 apoptosis signaling Treatment with Poly I:C (25 g/ml) for 4 h led to activation of caspase 8 (0.11 0.02 in controls vs. 0.161 0.03 OD 405 nm in cells treated with Poly I:C; p = 0.01). Preincubation of the cells with CsA at 1 M for 1 h prevented Poly I:C (25 g/ml for 30 min)-induced caspase 8 activation (0.11 0.02 in controls vs. 0.1 0.03 OD 405 nm in cells treated with Poly I:C; p = 0.6). CsA alone did not affect caspase 8 activity (Fig. 5a).

3.6. Cell and mitochondrial morphology Transmission electron microscopic examination revealed no major alterations in cellular or mitochondrial ultrastructure under poly I:C treatment (Fig. 6).

Fig. 4. NF-B activation is measured in cell extract 30 min after stimulation with Poly I:C (25 g/ml) pretreated with or without CsA at 1 M for 1 h, by a transcription factor Elisa (n = 4). Data represent mean SD. Statistical signicance between samples using unpaired t-test is indicated (*p b 0.05 vs. control and **p b 0.01 vs. control).

Fig. 5. a) Cell extracts were prepared from the unstimulated cells or cells incubated with Poly I:C (25 g/ml) for 4 h pretreated with or without CsA at 1 M for 1 h (n = 3). Caspase-8 activity (OD 450 nm) was determined using a caspase-8 colorimetric assay kit. Data represent mean SD. Statistical signicance between samples using unpaired t-test is indicated (*p b 0.05 vs. control). b) Western blot analysis of procaspase-3 and active cleaved caspases from HepG2 cells after incubation for 4 h with Poly I:C (25 g/ ml) or cobalt chloride (CoCl2, 1 mM and 3 mM; positive control) (n = 3).

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Fig. 6. HepG2 cells under control (a, c) and Poly I:C (25 g/ml, 4 h of incubation) (b, d) treatment conditions. Low-power views of cells (a, b) and high-power views of perinuclear mitochondria (c, d) are shown. No major ultrastructural alterations are visible under Poly I:C treatment.

complexes with transfecting reagents. This suggests that in our study, Poly I:C is internalized through endocytosis and recognized by TLR-3 rather than by intracellular cytoplasmic sensors melanoma differentiation-associated gene 5 (MDA-5) and retinoic acid inducible gene I (RIG-I) (Alexopoulou et al., 2001; De Miranda et al., 2009; Gitlin et al., 2006; Kato et al., 2005; Honda and Taniguchi, 2006). To assess TLRmediated signaling, we evaluated the activation of transcription factor NF-B in cultured hepatocytes following poly I:C stimulation. We demonstrated a poly I:C-induced activation of transcription factor NFB. It has been reported that one of the inhibitory effects of CsA is to interfere with IB degradation and to diminish the transcriptional activity of NF-B signaling (Marienfeld et al., 1997). We could also show that preincubation of the HepG2 cells with CsA did not prevent but diminished Poly I:C-induced NF-B activation. Our data suggest that the NF-b signalling pathway might not be directly involved in Poly I:C-induced mitochondrial dysfunction in cultured human hepatocytes. Another mechanism of CsA is to inhibit calcineurin phosphatase activity, therefore inhibiting the dephosphorylation and nuclear translocation of ubiquitous transcription factor NF-AT (nuclear factor of activated T-cells) (Flanagan et al., 1991; Liu et al., 1991). It has also been demonstrated that when human PBMCs and human Jurkat T cells are treated with Poly I:C, both NF-AT and NF-kB are activated in a concentration-dependent fashion (Kehoe et al., 2001). HepG2 cells express low amounts of endogeneous NF-AT (Hoey et al., 1995), and ectopic expression of NF-AT proteins transactivates NF-ATdriven transcription in this cell line in the absence of exogenous stimulation (Hoey et al., 1995). One potential limitation of our study is that we did not measure the activation of NF-AT after treatment of HepG2 cells with Poly I:C in the presence or absence of CsA. We have previously reported the lack of effect of Poly I:C on cellular respiration at 8 and 16 h (Regueira et al., 2009), and we decided to explore the effect of Poly I:C at earlier incubation time points. The current study is the rst showing that exposure of cultured hepatocytes to Poly I:C is associated with a decrease in cellular oxygen consumption at 2, 4 and 6 h of incubation. This effect was more prominent after 4 h of incubation (complexes I and IV) and was not present at 8 and 16 h of incubation (Regueira et al., 2009). Since reactive oxygen species can induce mitochondrial permeability transition (MPT), we examined the

effect of cyclosporine A, an MPT pore inhibitor (Kowaltowski et al., 2001), to determine whether oxidative stress is involved in mitochondrial dysfunction following poly I:C stimulation. We found that cyclosporine A prevented poly-I:C-induced reduction in complex I but not complex IV activity. The data indicate that MPT pore opening is one of the mechanisms contributing to the decrease in cellular respiration. Nevertheless, the protection by CsA was selective and partial because not all the complexes' activities were inhibited. The data indicate that mechanisms other than MPT pore opening must be involved. Such mechanisms may include pyruvate dehydrogenase inhibition (Vary, 1991) and poly(ADP-ribose) polymerase (PARP-1) activation (Goldfarb et al., 2002). Surprisingly CsA alone, at the dosage used, also induced a signicant reduction in complex IV-dependent respiration. Other reports on isolated mitochondria suggest that CsA may alter mitochondrial respiration in various tissues such as kidney (Lemmi et al., 1990), liver (Fournier et al., 1987) and skeletal muscles (Hokanson et al., 1995). Another downstream pathway of TLR-3 signaling is the apoptotic pathway, which results in the activation of caspase 8 (Kaiser and Offermann, 2005; Han et al., 2004). It was shown recently that caspase8 deciency in hepatocytes facilitates infection of the liver by Listeria monocytogenes, attenuates the hepatocyte proliferation after hepatectomy and prompts a chronic inammatory response to hepatectomy (Ben Moshe et al., 2007). In another study, Caspase 8 small interfering RNA prevented acute liver failure in mice (Zender et al., 2003). We showed that poly I:C induces activation of caspase 8 in cultured human hepatocytes. However, morphological examination revealed no major alterations in cellular or mitochondrial ultrastructure under poly I:C treatment, indicating no evidence of apoptosis. Nevertheless, caspase-8 is not always involved in cell death signaling, and non-apoptotic functions of caspase-8, involving embryonic development (Sakamaki et al., 2002), NF-B activation (Lemmers et al., 2007), and B cell proliferation (Beisner et al., 2005), have been reported. We showed that CsA blocks the Poly I:Cinduced activation of caspase 8. Caspase-8 is formed from the precursor protein pro-caspase-8 as a cleavage product and is predominantly localized in mitochondria, from which it is released upon apoptotic stimulation through a CsA-sensitive mitochondria permeability transition pore mechanism (Qin et al., 2001). To determine whether poly I:C induces activation of caspase-3 (activated by the upstream caspase-8) apoptosis signaling in HepG2

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cells, we examined processing/activation of caspase-3. Relatively little, if any, processing of procaspase-3 to active caspase-3 was detected in untreated cells or in cells treated with poly I:C. Thus, poly I:C used in our experiments did not induce key apoptosis signaling events in HepG2 cells. Previously we reported a lack of effect of poly I:C on mitochondrial respiration at 8 and 16 h (Regueira et al., 2009). We assume that activation of survival signaling, mitochondrial repair and biogenesis might be responsible for the lack of effect at later time points (Suliman et al., 2004, 2005). We speculate that poly-I:C-induced (2, 4 and 6 h incubation time) damage to the mitochondria might have been repaired through mitochondrial biogenesis. As discussed by others (Gunter and Pfeiffer, 1990), short, transient MPT pore opening does not create survival problems for either the mitochondrion or the cell. Reversible MPT pore opening might instead provide an energetically convenient way to eliminate undesired solutes from the mitochondrial matrix (Gunter and Pfeiffer, 1990). In summary, our data show that TLR-3 ligand (Poly I:C) induced a reduction in mitochondrial respiration in cultured human hepatocytes which was prevented partially by preincubation with cyclosporine A. Whether the relatively small effects of TLR3-mediated signaling on hepatocytes are physiologically relevant to contribute to liver dysfunction during sepsis has to be shown in further studies. Acknowledgements This study was supported by grants to SD and SMJ from the Stiftung fr die Forschung in Ansthesie und Intensivmedizin. We thank Ms. Sandra Nansoz for excellent technical assistance and Ms. Jeannie Wurz for excellent editorial assistance. References
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