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Department of Pathology and Center for Biodefense & Emerging Infectious Diseases, University of Texas Medical Branch, Galveston, TX, USA
Correspondence
Shu-Yuan Xiao
syxiao{at}utmb.edu
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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(TNF-) and the TNF receptor family. This study examined the roles of the TNF receptor-related extrinsic pathway and the Bcl-2 family-associated mitochondrial pathway in PTV-induced cell death. The effects of caspase inhibitors (caspIs) and TNF on cellular viability, virus replication, and morphological and biochemical changes in apoptosis were examined in HepG2 cells at different time points after infection with PTV (Adames strain). The results showed that caspIs dampened the virus-induced reduction in cellular viability, partially suppressed and delayed viral titres and antigen expression, and partially decreased the expression of apoptotic genes, caspase activities and DNA fragmentation. TNF treatment further decreased cellular viability after PTV infection and increased the level of apoptosis, whilst caspIs partially inhibited these effects. These findings indicate that TNF, caspase-8 and caspase-9 contribute to PTV-induced hepatocytic apoptosis and that additional mediators are probably also involved in this process. These mediators from different pathways correlated with one another and may be interlinked. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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; Fisher et al., 2003; Xiao et al., 2003; Nguyen & Blaho, 2007).
Punta Toro virus (PTV) is a member of the genus Phlebovirus, family Bunyaviridae (Nichol et al., 2005). In hamsters, PTV causes a fatal disease similar to the severe haemorrhagic fever caused by the phlebovirus Rift Valley fever virus in humans. We reported previously that PTV-induced apoptosis contributes to the death of hepatocytes and is responsible for severe liver damage (Fisher et al., 2003), which occurs in the absence of a secondary inflammatory cellular infiltration (Ding et al., 2005). Other investigators (Kang et al., 1999; Akhmatova et al., 2003; Markotic et al., 2003; Li et al., 2004, 2005; Klingström et al., 2006) have reported similar findings with other members of the family Bunyaviridae, in that hantaviruses trigger apoptosis directly or indirectly in Vero E6 (green monkey kidney), HEK293 (human embryonic kidney), epithelial and lymphopoietic cells. Cusi et al. (2005) demonstrated that a neuroadapted strain of Toscana virus (genus Phlebovirus) provoked neuronal apoptosis. Although these studies suggest that apoptosis plays an important role in the pathogenesis of hantavirus and phlebovirus infections, the molecular mechanisms involved remain to be elucidated.
Like many other types of cell, hepatocytes express ‘death’ receptors on the cell surface; this is perhaps related to the evolutionary pressure to facilitate the elimination of cells infected with hepatotropic viruses (Ghavami et al., 2005; Schattenberg & Galle, 2006). In contrast, the mitochondrial pathway is triggered by a variety of intracellular stressors such as DNA damage, growth factor deprivation, metabolic disturbances and detachment from matrix and/or surrounding cells (Sastre et al., 2007). These two pathways are not mutually exclusive but are closely interlinked, as the mitochondrial pathway is often required to amplify the relatively weaker death receptor-induced apoptotic signal in all cells, including hepatocytes (Yin & Ding, 2003). Many different mediators, such as Fas, human tumour necrosis factor- (TNF-) and members of the Bcl-2 family are also involved in apoptosis (Ghavami et al., 2005; Ewings et al., 2007; Hatano, 2007).
To find out which apoptotic pathways are involved in PTV-induced hepatocytic apoptosis, we investigated the potential factors related to PTV-induced apoptosis in HepG2 cells, including TNF- and caspases. Identification of the key steps involved may provide clues for the reduction of apoptosis; these steps in turn may be potential targets for future treatment of these diseases (Rust & Gores, 2000; Hatano, 2007).
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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).
Treatment with caspase inhibitors (caspIs) and TNF.
Caspase-9 inhibitor (casp9I, z-LEHD-FMK) or caspase-8 inhibitor (casp8I, z-IETD-FMK) (R&D Systems), at a concentration of 100 µM, was used to treat HepG2 cells 2 h before PTV infection to test their effects on virus-induced apoptosis. Recombinant TNF- (10 ng ml–1; Sigma) was used after the inoculation of PTV, with or without treatment with casp8I or casp9I. Cells were harvested and tested for cellular viability, virus titre, viral antigen, expression of genes related to apoptosis, phosphatidylserine translocation and DNA fragmentation at 24, 48 and 72 h post-infection (p.i.).
Virus titration.
The level of virus replication was measured by plaque assay. Briefly, after removal of the culture medium, confluent Vero E6 cell monolayers in 24-well plastic plates (Becton Dickinson Labware) were inoculated with 100 µl 10-fold serially diluted culture medium taken from infected HepG2 cells at the time points noted above. After adsorption at 37 °C and 5 % CO2 for 2 h, the inoculum was aspirated and each well was overlaid with a 1 ml mixture of minimal essential medium containing 2 % fetal calf serum (FCS) and 1 % carboxymethyl cellulose (Sigma). Culture medium from uninfected cells was used as a control at the same time points. Each sample was tested in duplicate. The plates were incubated at 37 °C with 5 % CO2 for 96 h, fixed with 10 % formalin overnight and then stained with a solution of 0.5 % crystal violet in PBS.
Indirect immunofluorescent assay (IFA).
For the IFA, HepG2 cells were grown in eight-well chamber slides (Nalge Nunc International), containing 104–105 cells per well. When the cell monolayer reached confluency, PTV was inoculated as indicated, with or without treatment with caspIs or TNF-. At 72 h p.i., the cells were fixed in cold acetone and prepared for IFA (Tesh, 1979). Briefly, 200 µl anti-PTV Adames antibody (mouse immune ascitic fluid) diluted 1 : 20 in PBS supplemented with 10 % FCS was added to each well for 45 min at 37 °C. After three washes in PBS, a secondary goat anti-mouse FITC-conjugated antibody (Sigma) diluted 1 : 50 in PBS and supplemented with 0.01 % Evans blue was added and incubated for 45 min at 37 °C. After three washes in PBS, the cells were dried and observed under an Olympus BX51 fluorescent microscope with an attached DP70 digital camera.
MTT assay.
At 24, 48 and 72 h p.i., cell viability was determined using a tetrazolium salt thiazolyl blue (MTT) (Sigma) assay as described previously (Ding et al., 2005).
RNA extraction, cDNA synthesis and real-time PCR.
At 24, 48 and 72 h p.i., total RNA was prepared from 106 infected or uninfected HepG2 cells, with or without different treatments, using TRIzol LS reagent (Invitrogen) and subjected to first-strand cDNA synthesis and real-time PCR using methods described previously (Ding et al., 2005). Expression of the genes for caspase-8, caspase-9, Bcl-2 and Bax was subsequently determined. The GAPDH gene was used as an internal control.
Annexin V–fluorescein staining.
HepG2 cells grown in eight-well chamber slides were inoculated with virus as described above. At 24, 48 and 72 h p.i., HepG2 cells from different treatment groups were harvested for the detection of phosphatidylserine translocation, according to a protocol described previously using Annexin V–fluorescein staining (Ding et al., 2005).
Terminal deoxynucleotidyl transferase-mediated dUTP nick end-labelling (TUNEL) assay.
At 24, 48 and 72 h p.i., a TUNEL assay was carried out as described previously (Xiao et al., 2001) to detect DNA fragmentation in HepG2 cells following different treatments.
Statistical analysis.
Cepheid Smart Cycle version 2.0b was used to analyse the raw data from real-time PCR. Experimentally determined standard curves and a comparative Ct method were used to analyse the raw data from each sample. SPSS version 12.0 was used to analyse pairwise comparisons using an independent-samples t-test and analysis of variance. A P value less than 0.05 was considered statistically significant.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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, cell viability in infected cells, as determined by an MTT assay, was decreased at 24 (P<0.05), 48 and 72 (P<0.01) h p.i. compared with the uninfected controls. When the infected cells were treated with casp8I, there was similar decrease at 24 (P<0.05) and 48 h (P<0.01), but the decrease disappeared at 72 h. When treated with casp9I, there was no significant change (P>0.05) between uninfected and infected cells. These results suggested that caspIs inhibit PTV-induced cell death at the later stages of infection. Cellular viability also decreased with TNF treatment after infection: casp8I inhibited this reduction at 72 h p.i. and casp9I inhibited it at 48 and 72 h p.i., implying that caspIs also dampen PTV/TNF-induced cell death in the later stages of infection.
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, casp8I decreased the virus titre at 48 and 72 h p.i. (P<0.01) and casp9I decreased the virus titre at 24 and 72 h p.i., compared with untreated groups (P<0.01). TNF treatment did not significantly change the virus titre (P>0.05), whilst caspIs treatment combined with TNF increased the virus titre at 24 h p.i. and then decreased it at 72 h p.i. (both P<0.01). These data indicated that caspI treatments decrease PTV titre in the later stages of infection.
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), the expression of viral antigen in HepG2 cells increased with time after infection and was strongest at 72 h p.i. Treatment with caspIs decreased the expression of viral antigen and infection was restricted to local areas. There was no obvious change with TNF treatment alone, but caspIs also suppressed viral antigen expression when combined with TNF treatment.
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). Expression of these genes increased significantly (P<0.05) at 72 h p.i. With casp8I treatment, expression of the caspase-9 and Bax genes increased significantly at 24 h p.i. (P<0.01) and at 72 h p.i. (P<0.05), respectively. Casp9I treatment increased the expression of caspase-8 at 24 h p.i. and decreased the expression of the Bcl-2 and Bax genes at 48 h p.i. (P<0.01). Expression of the four apoptotic genes also increased significantly (P<0.05) at 72 h p.i. with TNF treatment. The expression of caspase-8 and Bax increased significantly at 72 h p.i. (P<0.01 and P<0.05, respectively) and expression of caspase-9 and Bcl-2 increased at 48 h p.i. (P<0.01) following treatment with casp8I+TNF. All of these genes increased significantly at 48 h p.i. (P<0.01) and Bax gene expression also increased significantly at 72 h p.i. (P<0.05) following treatment with casp9I+TNF. These data indicated that individual caspIs can only partially suppress the virus-induced expression of apoptotic genes.
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, caspase-3 activity increased significantly (P<0.01) at different time points after infection. CaspIs suppressed this increase at 24 h p.i. and there was a significant increase (P<0.01) at 48 and 72 h p.i. for caspase-3 activity and at 48 h p.i. for caspase-8 activity (P<0.01) with caspIs treatments. The increase was also shown at different time points after treatment with TNF, whilst the addition of casp8I effectively dampened this increase, especially at 72 h p.i. These results suggested that caspases are involved in PTV-induced apoptosis and that caspase-8 or caspase-9 interacts with TNF during this process.
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). The number of annexin V-positive cells was significantly reduced in infected cells at 48 and 72 h p.i. (P<0.01) following casp8I or casp9I treatment, respectively. When treated with TNF, the number of annexin V-positive infected cells was still significantly increased at the different time points. Addition of Casp8I to TNF inhibited this increase, whilst addition of casp9I partially inhibited it. The number of annexin V-positive cells was still significantly (P<0.01) increased at 48 h p.i. following casp9I+TNF treatment.
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). In untreated cells, the number of positive cells was increased significantly (P<0.01) at 48 and 72 h p.i. It was decreased by treatment with Casp8I at 48 h p.i. and by treatment with casp9I at 24 and 48 h p.i. When infected cells were treated with TNF, there were significant increases in the number of TUNEL-positive cells at 48 (P<0.01) and 72 (P<0.05) h p.i. The addition of caspIs did not inhibit this increase compared with the TNF-treated group.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). However, the apoptotic pathways involved and the roles of key members such as TNF-, caspase-8 and caspase-9 (Budihardjo et al., 1999; Earnshaw, 1999; Hatano, 2007) were not examined systemically in PTV-induced hepatocytic injuries. In this study, it was shown that caspIs delayed and suppressed the cytopathic effect induced by PTV. This was confirmed by a cellular viability analysis, demonstrating that casp9I reversed the virus-induced reduction in cell viability at different time points, and at 72 h p.i. with casp8I treatment (Fig. 1
). This was accompanied by reduced virus replication, as shown by a significant (P<0.01) decrease in virus titre at 24 and 72 h p.i. with casp9I treatment, and at 48 and 72 h p.i. with casp8I treatment (Fig. 2), and by the evident reduction in viral antigen expression at 72 h p.i. with caspI treatment (Fig. 3). These data indicated that caspase-8 and caspase-9 are both mediators of PTV-induced apoptosis. Furthermore, casp8I and casp9I suppressed gene expression of the caspases and Bcl-2 family members (Fig. 4) and decreased caspase-8 and caspase-9 activities at 72 h p.i. (Fig. 5). Inhibition of phosphatidylserine translocation (Fig. 6) and DNA fragmentation were also noted in the early stages of infection. However, as complete inhibition of virus-induced apoptosis was not reached with any of the treatments (Figs 5 and 7), it is likely that caspase-8 and caspase-9 are not the only ‘players’ in this process and that other apoptotic pathways (besides caspase-8-dependent extrinsic and caspase-9-dependent intrinsic mitochondrial pathways) may also be involved in PTV-induced apoptosis.
When treated with casp9I, virus titres increased significantly (P<0.01) at 48 h p.i. (Fig. 2). This is an unusual observation. One possible explanation is that the virus titre at 24 h p.i. with casp9I treatment was too low to permit PTV to proliferate and spread efficiently, so the viruses modulated or changed the signal transduction of host cells in order to survive, as a defence mechanism of the virus (Boya et al., 2004). Furthermore, there was a significant increase in the expression of caspase-9 after treatment with casp8I and in the expression of caspase-8 after the treatment with casp9I (Fig. 4), suggesting that caspase-8 and caspase-9 interact with each other.
Recombinant TNF- has an anti-tumour cell effect (Sugarman et al., 1985) and promotes cell death by inducing the activation of the cysteine proteases caspase-8 and caspase-3 (Nicholson & Thornberry, 1997). It has been implicated in the pathogenesis of viruses causing haemorrhagic fever with renal syndrome and hantavirus pulmonary syndrome (Temonen et al., 1993; Mori et al., 1999). In this study, TNF increased the expression of caspase-8, even when combined with casp8I (P<0.01), and decreased the expression of Bcl-2 (P<0.01), an anti-apoptotic protein (Fig. 4). With TNF treatment, apoptosis effector caspase-3 and the apoptosis initiators caspase-8 and caspase-9 (Fig. 5), as well as apoptosis markers (phosphatidylserine translocation and DNA fragmentation), were all significantly increased (P<0.05) at some points after PTV infection compared with the untreated group (Figs 6 and 7). These results confirm that TNF- participates in PTV-induced apoptosis, possibly by inducing activation of the cysteine proteases. Surprisingly, caspIs did not inhibit the effects caused by TNF treatment. These results were consistent with results reported by others (Nicholson & Thornberry, 1997; Hatano, 2007) and indicate that other mediators, in addition to TNF-, caspase-8 and caspase-9, are also involved in PTV-induced apoptosis. These mediators may enhance or amplify the cross-talk between intrinsic and extrinsic apoptotic pathways, especially when one is blocked or inhibited.
In summary, the findings of the present study provide insight into the relationship at the molecular level between phleboviruses and the apoptotic process of infected host cells, and into the pathogenesis of phlebovirus-induced diseases.
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ACKNOWLEDGEMENTS |
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Received 27 February 2008;
accepted 8 May 2008.
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