Elsevier

Mitochondrion

Volume 11, Issue 1, January 2011, Pages 83-88
Mitochondrion

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

https://doi.org/10.1016/j.mito.2010.07.010Get rights and content

Abstract

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 amplifies the systemic hyperinflammatory 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 — polyinosine–polycytidylic 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-κB, and the mitochondrial dysfunction was accompanied by caspase-8 but not caspase-3 activation and by no major alterations in cellular or mitochondrial ultrastructure.

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 significantly decreases sepsis-induced mortality in mice (Cavassani et al., 2008). TLR-3 ligand amplifies the systemic hyperinflammatory response observed during sepsis (Doughty et al., 2006). The data indicated that both the interferon α/β and NF-κB pathways (actived by treatment of mice with TLR-3 ligand) were critical for potentiation of the inflammatory 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 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-κB 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., 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.

Section snippets

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).

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 μg/ml) or cobalt

Cellular oxygen consumption after TLR-3-agonist stimulation

Representative diagram of measurement of respiration rates in permeabilized HepG2 cells at an experimental density of 1 × 106/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 flux (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

Discussion

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

Acknowledgements

This study was supported by grants to SD and SMJ from the “Stiftung für die Forschung in Anästhesie und Intensivmedizin”. We thank Ms. Sandra Nansoz for excellent technical assistance and Ms. Jeannie Wurz for excellent editorial assistance.

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    Present address: Institute of Functional and Applied Anatomy, Hannover Medical School, D-30625 Hannover, Germany.

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