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Summary

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. References

The group B coxsackieviruses are single-stranded RNA viruses that have been implicated in viral myocarditis. Viral infection of the myocardium, as well as the associated inflammatory response are important determinants of the virus-associated myocardial damage. Although these viruses are known as cytopathic viruses that cause death of the host cell, their viral RNA has been shown to persist in cardiac muscle contributing to a chronic inflammatory cardiomyopathy. Thus, it is essential that we understand the mechanism by which Coxasckie B viruses (CBVs) trigger this inflammatory response. In this study we investigated the involvement of Toll-like receptors (TLRs) in the recognition of CBV virions as well as CBV single-stranded RNA. Here we report that the CBV-induced inflammatory response is mediated through TLR8 and to a lesser extent through TLR7.


Introduction

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. References

Viral infections of the heart are important causes of morbidity and mortality in adults and children. Dilated cardiomyopathy (DCM) is the major reason for cardiac transplantation in the USA and Europe (Manolio et al., 1992). DCM appears to occur as a result of acute or chronic viral myocarditis (Noren et al., 1977; Woodruff, 1980; O’Connell, 1987; Chow et al., 1992; Dec and Fuster, 1994), either through persistence of the virus (Chow et al., 1992) or to an autoimmune phenomenon occurring after virus exposure (Huber, 1997). Viral myocarditis has been demonstrated to be induced primarily by at least two types of viruses, enteroviruses and adenoviruses. The primary viruses that have been aetiologically linked to myocarditis and DCM are the human enteroviruses. Studies have demonstrated the persistence of enteroviral infection of the myocardium in patients presenting myocarditis (Bowles et al., 1986; 1989; Kandolf et al., 1987; 1991). Among the enteroviruses most commonly associated with these diseases are the Coxasckie B viruses (CBV), and in particular Coxsackie B3 and B5 viruses (Grist and Reid, 1993).

Even though Coxsackie B viruses have been implicated in the development of viral myocarditis the exact mechanisms that result in the onset of the disease still remain unknown. Myocardial tissue damage may either result from viral infections or from a disregulated immune response. In addition, the role of inflammatory cytokines in viral-induced myocarditis has not been well defined. It has been suggested that the cytokines produced in response to the virus might play an important role in the pathogenesis of CBVs (Gluck et al., 2001). In particular, persistence of viral genomes in the myocardium of infected mice has been associated with high levels of cytokines, which are believed to contribute to the pathological changes of CBV3-induced myocardial pathogenesis (Neumann et al., 1993; Hober et al., 1996; Gluck et al., 2001).

Toll-like receptors (TLRs) are a conserved family of receptors that act as the first line of defence of the innate immune system against microbial pathogens. It is emerging that TLRs are part of the inflammatory response against viruses and viral ligands. In particular, TLR2, TLR3, TLR4, TLR8 and TLR9 have been shown to play a role in the cytokine response against various viruses, including herpes simplex virus, cytomegalovirus, respiratory syncytial virus, mouse mammary tumour virus and measles virus (Kurt-Jones et al., 2000; 2004; Bieback et al., 2002; Rassa et al., 2002; Compton et al., 2003; Tabeta et al., 2004). In the case of CBVs, TLR4 has been shown to be implicated in the innate immune response against them (Frantz et al., 1999; Birks et al., 2004; Satoh et al., 2004; Triantafilou and Triantafilou, 2004). However, recently TLR7 and TLR8 has been shown to mediate anti-viral responses by means of recognizing single-stranded RNA (ssRNA) (Diebold et al., 2004; Heil et al., 2004; Lund et al., 2004).

In this study we aimed to elucidate the role of TLRs in coxsackievirus group B-induced cardiac inflammation. Using human embryonic kidney (HEK)-transfected cell lines, we demonstrate that TLR7 and TLR8 are involved in the inflammatory responses against CBV3 and CBV5. TLR7 and TLR8 seem to recognize CBV ssRNA within endosomes. Using confocal microscopy we demonstrate that upon viral internalization, the viral RNA is released in acidified endosomal compartments, and triggers a cytokine response through TLR8 and to a lesser extent through TLR7.

Results

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. References

CBV3 and CBV5 activation of human cardiac cells

Viral myocarditis has been shown to have two different phases: an early inflammatory phase and a chronic phase, which later progresses to myocardial failure. The early stage is characterized by the presence of inflammatory cells in the myocardium, as well as the presence of elevated levels of inflammatory cytokines (Yamada et al., 1994). Several studies have suggested that these proinflammatory cytokines might have a negative effect on cardiac function (Sobotka et al., 1990; Finkel et al., 1992; Hosenpud, 1993). Thus, initially we investigated whether CBV3 could induce the production of proinflammatory cytokines from human cardiac cells.

Human cardiac cells were incubated with CBV3 virions for different time periods. It was shown that CBV3 can induce IL-6, IFN-β and to a lesser extent TNF-α in human cardiac cells (Fig. 1, black bar charts).

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Figure 1. CBV3 activation of human cardiac cells. Human cardiac cells were incubated with CBV3 virions (1 × 103 PFU ml−1) (black bar charts) or UV-inactivated CBV3 (white bar charts) for 1 h. The supernatants were harvested and assayed for cytokine contents using the Cytometric Bead Array (CBA) system (Becton Dickinson). Fluorescence was detected using a FACSCalibur (Becton Dickinson). The data presented are the mean of three independent experiments.

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In order to further investigate whether CBV3 infection of cardiac cells is required for the induction of cytokines, we incubated human cardiac cells with UV-inactivated CBV3 particles. These particles are unable to infect cells. It was shown that these particles were able to minimally stimulate cytokine production (Fig. 1, white bar charts), thus demonstrating that the initial contact between the viral particle and the cells is not sufficient in order to stimulate the proinflammatory cytokine production. It seems that the virus has to internalize in order to fully activate cardiac cells. Similar results were obtained with CBV5.

TLR expression on human cardiac cells

Several TLRs have been implicated in the innate recognition of viruses, thus we investigated the total TLR expression on cardiac cells before and after infection by CBV3.

We found that cardiac cells expressed minimal levels of all TLRs that we tested (TLR1–8) before CBV3 infection (Fig. 2, grey bar charts). Interestingly, it was shown that the expression of TLR7 and TLR8 increased upon infection with CBV3 virions (Fig. 2, black bar charts), whereas in the presence of UV-CBV3 virions TLR7 and TLR8 expression remained the same (Fig. 2, white bar charts). As TLR7 and TLR8 have been shown to be able to recognize ssRNA (Heil et al., 2004; Lund et al., 2004), we decided to test whether viral ssRNA will be able to trigger innate immune responses. Upon stimulation of human cardiac cells with CBV3 single-stranded genomic RNA, we found that, similarly to CBV3 virions, TLR7 and TLR8 expression was upregulated (Fig. 2, striped bar charts). Similar results were obtained with CBV5. Thus, our data suggest that the innate immune system can sense the viral RNA and activate human cardiac cells in response to CVBs.

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Figure 2. TLR expression in human cardiac cells. Human cardiac cells were either not stimulated (grey bar charts) or stimulated with CBV3 virions (1 × 103 PFU ml−1) (black bar charts), UV-inactivated CBV3 (white bar charts) or 20 µg ml−1 CBV3 ssRNA (striped bar charts) for 2 h. The cells were fixed and permeabilized, followed by antibody staining against the particular TLR molecule, and incubation with the appropriate secondary antibody was conjugated to FITC. Fluorescence was detected using a FACSCalibur (Becton Dickinson). The data presented are the mean of three independent experiments.

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CBV-induced activation is mainly TLR7- and TLR8-dependent

In order to investigate which TLRs might play a role in CBV3-induced activation of human cardiac cells, we utilized transfected cell lines. HEK cells transfected with either TLR2, TLR3, TLR4/MD2, TLR7 or TLR8 were utilized. HEK cells, which do not express TLRs, were found not to be able to produce IL-6, TNF-α or IFN-β in response to CBV3 (Fig. 3). Similarly CBV3 did not trigger cytokine production in HEK cells transfected with TLR2 or TLR3. HEK-TLR4 cells were shown to produce small amounts of IL-6, TNF-α and IFN-β in response to CBV3 and UV-CBV3 (Fig. 3). On the contrary, HEK cells transfected with either TLR7 or TLR8 produced IL-6, TNF-α and IFN-β after incubation with CBV3 virions. Interestingly HEK cells transfected with TLR8 had the highest IL-6 response against CBV3 (Fig. 3, black bar charts), thus suggesting that CBV3 induced activation in mediated mainly through TLR8. Control cultures were stimulated with known TLR2, TLR3 or TLR4 ligands (data not shown).

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Figure 3. TLR7- and TLR8-dependent cytokine secretion in response to CBV3. HEK-293 cells transfected with TLR2, TLR3, TLR4, TLR7 and TLR8 were incubated with either CBV3 virions (1 × 103 PFU ml−1) (black bar charts), UV-CBV3 (white bar charts) or 20 µg ml−1 CBV3 ssRNA (striped bar charts) for 2 h. The supernatants were harvested and assayed for cytokine (IL-6, TNF-α and IFN-β) content using the Cytometric Bead Array (CBA) system (Becton Dickinson). Fluorescence was detected using a FACSCalibur (Becton Dickinson). The data represent the mean of three independent experiments.

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In order to determine whether the induction of cytokines was dependent on replication competence, we stimulated the transfected cell lines with UV-inactivated CBV3 virions. We found that UV-inactivated CBV3 virions were able to stimulate some IL-6, TNF-α and IFN-β production from HEK-TLR4 cells, but were not able to stimulate the production of cytokines in HEK-TLR7 or HEK-TLR8 cells (Fig. 3, white bar charts), thus demonstrating that infection of cells by CBV3 is required in order to activate the innate immune response. HEK-TLR7 and HEK-TLR8 responded to CBV3 ssRNA, whereas HEK-TLR4 did not (Fig. 3, striped bar charts). In order to make sure that ssRNA was indeed the trigger for HEK-TLR7 and HEL-TLR8 cytokine production, HEK cells transfected with either TLR7 or TLR8 were stimulated with 20 µg ml−1 synthetic polyU RNA. The cytokine response triggered by polyU RNA was identical to that triggered by CBV3 ssRNA, demonstrating that ssRNA is the trigger in HEK-TLR7 and HEK-TLR8 cells (data not shown). Thus, it seems that CBV3 particles can in some part be recognized on the cell surface by TLR4, but they mainly trigger the innate immune response by internalizing and releasing ssRNA within the host cell.

NF-κB transcriptional responses to CBVs are mediated by TLR7 and TLR8

Toll-like receptors seem to act upstream of NF-κB activation. TLR signalling pathways have been shown to ultimately result in the release of NF-κB from its endogenous inhibitor (Akira, 2001) and subsequent nuclear translocation that leads to the transcription of inflammatory cytokines.

In order to determine whether the CBV3-induced TLR7 and TLR8 activation that we observed leads to NF-κB-driven transcription response, we utilized HEK cells transfected with either TLR2, TLR3, TLR4, TLR7 or TLR8 and an NF-κB luciferase reporter gene. Cells were stimulated as indicated and after 6 h of stimulation, the cells were lysed in passive lysis buffer (Promega). Luciferase activity was measured using a plate reader luminometer. Similar to our previous findings, it was shown that CBV3 virions were able to induce some NF-κB activation in HEK-TLR4 cells. Whereas CBV3 virions (Fig. 4, black bar charts) as well as CBV3 ssRNA (Fig. 4, striped bar charts) were able to induce NF-κB activation only in cells that expressed either TLR8 or to a lesser extent TLR7.

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Figure 4. TLR7- and TLR8-dependent NF-κB activation in response to CBV3. HEK293 cells transfected with either TLR2, TLR3, TLR4, TLR7 or TLR8 and a luciferase reporter gene were incubated with either no stimulus (grey bar charts), CBV3 virions (1 × 103 PFU ml−1) (black bar charts), UV-inactivated CBV3 (white bar charts) or 20 µg ml−1 CBV3 ssRNA (striped bar charts). After stimulation, the cells were lysed and analysed for luciferase activity. The data shown represent a mean of three independent experiments.

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Inhibition of CBV activation of human cardiac cells by silencing TLR8 and to a lesser extent TLR7

As we had already demonstrated the importance of TLR7 and TLR8 in CBV3-mediated activation in transfected cell lines, we investigated whether TLR7 and TLR8 were also important for triggering the inflammatory response in human cardiac cells.

In order to determine the role of TLR7 and TLR8 in CBV3 ssRNA recognition, we used RNA interference (siRNA) to knock down the expression of TLR7 and TLR8 in primary human cardiac cells. Transfection with synthetic TLR7- or TLR8-specific plasmid encoding small interfering RNA (psiRNA) resulted in 60% decrease in TLR8 and 50% decrease in TLR7 expression as determined by Western blotting (Fig. 5A). Control transfections of human cardiac cells with the psiRNA vector did not affect TLR7 or TLR8 expression.

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Figure 5. Inhibition of CBV3 activation of human cardiac cells by silencing TLR8 and TLR7. RNA interference resulted in decreased expression of TLR7 and TLR8 as determined by Western blotting (A). Following RNA interference, human cardiac cells were not stimulated (grey bar charts), or incubated with either CBV3 virions (black bar charts), UV-CBV3 (white bar charts) or 20 µg ml−1 CBV3 ssRNA (striped bar charts) for 2 h (B). The supernatants were harvested and assayed for cytokine secretion using the Cytometric Bead Array (CBA) system (Becton Dickinson). Fluorescence was detected using a FACSCalibur (Becton Dickinson). The data represent the mean of three independent experiments.

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Following RNA interference, human cardiac cells were incubated with CBV3 virions (Fig. 5B, black bar charts), UV-CBV3 (Fig. 5B, white bar charts) or CBV3 ssRNA (Fig. 5B, striped bar charts). Cytokine assays were performed after the designated incubation times. It was shown that silencing of TLR8 inhibited CBV3 or CBV3 ssRNA-induced cellular activation (Fig. 5B), thus suggesting the importance of TLR8 in CBV3-mediated activation of human cardiac cells. Although silencing of TLR7 also inhibited CBV3-induced cellular activation, it was to a much lesser extent, thus suggesting that innate immune responses against CBV3 are triggered mainly through TLR8. Control experiments were performed by employing psiRNA technology in order to silence TLR2. Transfection with synthetic TLR2-specific psiRNA resulted in 60% decrease in TLR2 expression as determined by luciferase activity and Western blotting. Following RNA interference for TLR2, human cardiac cells were incubated with CBV3, UV-CVB3 and CBV3 ssRNA. Silencing of TLR2 did not affect the production of IL-6 in response to CBV3 and CBV3 ssRNA, thus suggesting that the inhibition observed when TLR7 and TLR8 were silenced was specific. TNF-α and INF-β were induced in the same way (data not shown). Similar results were obtained with CBV5.

Association of CBV ssRNA and TLR7/TLR8 within endosomes

In order to examine the mechanism by which ssRNA from CBV3 was recognized via TLR7 and TLR8, we labelled the virus RNA with Alexa 488. Using confocal microscopy we investigated the uptake and trafficking of CBV3 ssRNA in human cardiac cells, in order to determine whether it moves to the same compartment in which TLR7 and TLR8 reside. Although picornavirus naked RNA has been shown in previous studies to be infectious (at a much lower percentage than the complete virions) to cells, we also used cationic lipids to facilitate uptake of RNA as it has been shown that RNA complexed with Dotap can be easily uptaken by a cell (Boczkowski et al., 1996).

To visualize endocytic organelles within the cells, we labelled lysosomes with lysotracker TRITC, while early endosomes were labelled with Cy3-conjugated EEA-1 serum. Our results show that within 15 min after infection, Alexa 488-ssRNA colocalized with TLR8 into early endosomes (Fig. 6). Similar results were obtained when we investigated TLR7 and ssRNA colocalization (Fig. 7), thus verifying that TLR7 and TLR8 recognize viral ssRNA within endosomal compartments.

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Figure 6. CBV3 ssRNA and TLR7 colocalize in endosomes. Intracellular distribution of CBV3 ssRNA and TLR7. CBV3 ssRNA (20 µg ml−1) (green) and TLR7 (blue) colocalized within endosomes (red) within 15 min. Images were collected using a Zeiss 510 META confocal microscope. The merged image shows extensive overlay of areas positive for CBV3 ssRNA, TLR7 and endosomes. Scale bar, 10 µm.

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Figure 7. CBV3 ssRNA and TLR8 colocalize in endosomes. Intracellular distribution of CBV3 ssRNA and TLR8. CBV3 ssRNA (20 µg ml−1) (green) and TLR8 (blue) colocalized within endosomes (red) within 15 min. Images were collected using a Zeiss 510 META confocal microscope. The merged image shows extensive overlay of areas positive for CBV3 ssRNA, TLR8 and endosomes. Scale bar, 10 µm.

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Recruitment of MyD88 to endosomal compartments

In order to investigate whether signalling was triggered once the viral ssRNA reached the endosomal compartment, we investigated the spatial behaviour of the adaptor protein MyD88. MyD88 was found to be expressed ubiquitously in the cytoplasm of resting human cardiac cells (Fig. 8). Fifteen minutes after infection, we observed a rapid redistribution of MyD88 to endosomal compartments (Fig. 8). Extensive colocalization of fluorescently labelled TLR7 or TLR8 and MyD88 was observed in these compartments within 15 min of addition of Alexa 488-ssRNA (Fig. 9). These results suggest that both TLR7 and TLR8 are localized in endosomes where they are able to recognize ssRNA that has been released from the viral capsid. This recognition seems to lead to signalling, as suggested by the recruitment of MyD88, and subsequently to a proinflammatory response.

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Figure 8. CBV3 ssRNA recruits MyD88 in endosomes. Human cardiac cells were either not stimulated (left) or stimulated with CBV3 ssRNA (20 µg ml−1) (right) and imaged using a Zeiss 510 META confocal microscope. Intracellular MyD88 was stained using a polyclonal antibody directly labelled with FITC. Endosomes were labelled using EEA1 and Rhodamine-labelled swine anti-rabbit secondary, Topro was used to label the nucleus of the cells. Merged images showing extensive overlay of areas positive for MyD88 and EEA1 are seen as yellow. Scale bar, 10 µm.

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image

Figure 9. CBV3 ssRNA colocalizes with MyD88 and TLR8 in endosomes. Human cardiac cells were incubated with Alexa-488-CBV3 ssRNA (20 µg ml−1) and imaged using a Zeiss 510 META confocal microscope. Intracellular MyD88 was stained using a polyclonal antibody directly labelled with TRITC. TLR8 was labelled using TLR8-specific polyclonal serum and Cy5-labelled swine anti-rabbit secondary. Merged images showing extensive overlay of areas positive for MyD88, CBV3 ssRNA and TLR8 are seen as white. Scale bar, 10 µm.

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Discussion

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. References

The group B coxsackieviruses are enteroviruses of the family Picornaviridae that cause a variety of human diseases, most notably life-threatening conditions such as aseptic meningitis, pancreatitis and myocarditis (Pallansch, 1997). Coxsackieviruses consist of a naked icodahedral capsid enclosing a single-stranded, messenger-sense, polyadenylated RNA genome of approximately 7400 nucleotides in length. While coxsackieviruses are best known as cytopathic viruses that cause death of the host cell, viral RNA has been shown to persist in skeletal muscle, cardiac muscle, spleen and lymph nodes (Tam et al., 1991; 1994; Klingel et al., 1995). The persistence of viral RNA within the host is thought to contribute to the chronic inflammatory response against the virus (Tam et al., 1994).

In the case of viral myocarditis, it has been shown that both viral infection of the myocardium (Chow et al., 1992) and the associated inflammatory response (Craighead et al., 1990) are important determinants of the degree of virus-associated myocardial damage. The persistence of viral RNA in the cardiac muscle is believed to be associated with the inflammatory response observed. Thus, it is imperative to elucidate how the viral RNA can induce an inflammatory response, if we are to understand how the virus causes myocardial damage.

Initially we investigated whether CBVs can trigger the secretion of proinflammatory cytokines in human cardiac cells. Our experiments suggest that CBV3 and CBV5 are capable of inducing the secretion of proinflammatory cytokines, which include TNF-α and to a greater extent IFN-β and IL-6, within the first 2 h of its interaction with the host. This is in good agreement with recent findings by Fairweather et al. (2004) in mice. Thus, we proceeded to elucidate the mechanism by which CBVs can induce a cytokine response in human cardiac cells. As TLRs have recently been emerging as the key receptors for sensing viruses and viral DNA or RNA, we investigated whether TLRs were involved in the cytokine production in response to CBV3. In order to examine which if any, of the TLR molecules are involved, we utilized HEK293 cells transfected with different TLRs. It was shown that CBV virions were able to activate only cells transfected with either TLR7 or TLR8 and to a lesser extent TLR4. TLR8 seems to be a more ‘efficient sensor’ of CBV3 ssRNA, which is in agreement with Heil et al. (2004) who have demonstrated that murine TLR7 and human TLR8 mediate recognition of ssRNA. The ability to induce an inflammatory response via TLR7 and TLR8 seemed to require the internalization of CBVs, or the successful infection of human cardiac cells, as UV-inactivated CBV3 virions (which are incapable of infecting cells) were not able to trigger the exact same response. In contrast, only CBV3 and UV-CBV3 virions were able to trigger a small inflammatory response via TLR4, thus suggesting that the innate recognition of CBV3 in the early stages of attachment to the host cells is via TLR4, but once the virus has infected the cells TLR7 and to a greater extent TLR8 are the main initiators of the inflammatory response. This seems to be consistent with the fact that TLR7 and TLR8 are mostly expressed intracellular, and thus the virus must internalize in order to interact with these receptors.

As coxsackieviruses are ssRNA viruses, we proceeded to test whether the viral RNA was able to stimulate a cytokine production. It was shown that CBV3 ssRNA could trigger a cytokine response only in cells, which expressed TLR7 or TLR8. When we investigated the intracellular distribution of CBV3 ssRNA, we found that it was targeted to early endosomes within 15 min after infection. TLR7 and TLR8 were found to colocalize with CBV3 ssRNA within endosomes. This interaction seemed to trigger the recruitment of signalling molecules, such as MyD88 to early endosomes. MyD88 was found to be constitutively expressed throughout the cytoplasm in unstimulated cells, but was rapidly redistributed into endosomes as soon as the cells were infected with CBV3.

Overall our data suggest that the inflammatory response triggered by CBVs in cardiac cells is mediated by the synergic activation of multiple TLRs. It seems that a small part of the inflammatory response is triggered in the initial stages of the virus attachment to the cell surface and is mediated via TLR4. This is in good agreement with recent findings that link TLR4 with CBV-associated myocarditis (Frantz et al., 1999; Fairweather et al., 2003; Birks et al., 2004; Satoh et al., 2004). In addition, it seems that the main inflammatory response is triggered once CBVs have internalized and released their RNA into endosomes, where TLR7 and TLR8 reside. It seems that CBV RNA replication is essential for TLR7- and TLR8-mediated immune responses. This is in agreement with several studies which have reported that enteroviral RNA replication plays an important role in immune responses in human dilated cardiomyopathy (Pauschinger et al., 1999; Fujioka et al., 2000; Satoh et al., 2004). In infected individuals, TLR7 and TLR8 must be continuously activated by the presence of viral ssRNA during enteroviral replication, whereas upon cell lysis TLR4 must be triggering an immune response against new virions. The synergic inflammatory response of TLR4, TLR7 and TLR8 seem to produce a chronic inflammatory response against the virus, which could eventually lead to irreversible myocardial injury. The current study sheds new light into the mechanisms by which CBVs cause chronic inflammation of the myocardium, and might help us find new therapeutic targets for viral-induced cardiac inflammation.

Experimental procedures

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. References

Cells

HEK293 cells transfected with either TLR2, TLR3 or TLR4 were kindly provided by Professor Douglas Golenbock (University of Massachusetts Medical School, Worcester, USA). Stable transfections of HEK/TLR1, HEK/TLR7 and HEK/TLR8 cells with puno-TLR1, puno-TLR7 or puno-TLR8 plasmids (Invivogen) were performed using Lipofectamine 2000 according to the manufacturer's recommendations. Positive selection by fluorescence-activated cell sorting was performed. Clonal cell lines were obtained by limiting dilution.

Transfected cell lines were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum, 0.5 units ml−1 penicillin, 0.5 µg ml−1 streptomycin, 400 µg ml−1 G418 and 10 µg ml−1 Ciprofloxacin for HEK/TLR2, HEK/TLR3 and HEK/TLR4 and 10 µg ml−1 blasticidin for HEK/TLR1, HEK/TLR7 and HEK/TLR8.

Primary human aortic muscle cells isolated from a healthy, 30-year-old female were purchased from (Promocell, Heidelberg, Germany) and maintained in muscle cell growth medium. The cell cultures are produced at PromoCell's cell culture facility from normal human tissue obtained from surgical operations. The cells are isolated according to referenced procedures. Each isolate undergoes extensive testing for the presence of specific cardiac cell markers as well as the absence of specific cell markers.

Viruses

Prototype strains of coxsackievirus B3 (CBV3) and coxsackievirus B5 (CBV5) were obtained from the American Type culture collection (ATCC). Viral genomic RNA was isolated from purified virus using TRIzol Reagents (Invitrogen) according to manufacturer's instructions. Viral purified RNA was used to stimulate cells at 20 µg ml−1.

Materials

All fine chemicals were obtained from Sigma (UK). TLR1-, TLR3-, TLR5-, TLR6-, TLR7-, TLR8- and MyD88-specific polyclonal antibodies were obtained from Autogen Bioclear (UK). TL2.1, TLR2-specific monoclonal antibody (mAb) and HTA125, TLR4-specific mAb were obtained from HyCult (Netherlands). Lysotraker Red DND-99 and Alexa 488-Ulysis reagent were obtained from Molecular Probes (Cambridge Biosciences, Cambridge, UK). CBV3 ssRNA was labelled with Alexa-488-Ulysis reagent according to the manufacturer's instructions. EEA1 goat polyconal serum specific for early endosomes was obtained from Autogen Bioclear (UK).

Flow cytometric determination of TLR expression

In order to investigate TLR expression before and after CBV infection, human cardiac cells were either infected with CBVs for 2 h or not, before fixation with 4% paraformaldehyde. The cells were subsequently washed and permeabilized using PBS/0.02% BSA/0.02% Saponin. After permeabilization, the cells were incubated with antibodies against different TLRs and the appropriate secondaries conjugated to FITC. Appropriate isotype controls were also used. The cells were washed twice in PBS/0.02% BSA/0.02% Saponin and resuspended in 500 µl of PBS. Fluorescence was detected using a FACSCalibur counting 10 000 cells not gated.

Cytokine assays

Human primary cardiac cells were stimulated with no stimulus or with CBV3 or CBV5 virions [1 × 103 plaque forming units (PFU) ml−1]. The cultures were incubated for the designated times. The supernatants were collected and frozen until the cytokine assays were performed. The Becton Dickinson bead array system was used in order to determine the level of multiple cytokines at the same time.

Luciferase reporter assays for NF-κB activation

HEK293 cells transfected with either TLR1, TLR2, TLR3, TLR4, TLR7 or TLR8 were seeded into 96-well plates. The following day, the cells were transiently transfected with an NF-κB luciferase reporter gene using lipofectamine 2000 (Invitrogen, UK) according to the manufacturer's instructions. The next day the cells were stimulated as indicated and after 6 h of stimulation, the cells were lysed in passive lysis buffer (Promega). Luciferase activity was measured using a plate reader luminometer.

Western blotting

Human primary cardiac cells (1 000 000) before and after silencing were lysed in 500 µl of lysis buffer. Lysates (20 µl per sample) were analysed by SDS-PAGE and transferred onto a nitrocellulose filter (Schleicher-Schuell, Germany) or Immobilon P membranes (Millipore) for 1 h at 220 mA in the presence of transfer buffer (20 mM Tris-acetate, 0.1% SDS, 20% isopropanol, pH 8.3). After transfer, the membrane was blocked for 1 h in blocking solution (5% low-fat dried milk dissolved in PBS-T) and washed with PBS-T (two rinses, a 15 min wash, and two 10 min washes). Membranes were probed with the appropriate dilution of primary antibody for 1 h followed by washing with PBS-T. Membranes were incubated with horseradish peroxidase (HRP) conjugated to either swine anti-rabbit Ig (1:4000), donkey anti-goat Ig or rabbit anti-mouse Ig for 1 h. After extensive washing with PBS-T, the antigen was visualized using the ECL procedure (Amersham Pharmacia) according to the manufacturer's instructions.

RNA interference

RNA interference was used in order to silence the TLR7 and TLR8 genes. Four psiRNA clones were generated using the psiRNA-h7SK vector from Invitrogen for TLR7; the most efficient was against the sequence 5′-GGGTATCAGCGTCTAATATCA-3′. For TLR8 the following sequence was targeted: 5′-GACCAACTT CGATACCTAAA-3′. For TLR2, the following sequence was targeted: 5′-GTCAATTCAGAACGTAAGTCA-3′. Human primary cardiac cells (1 × 105) were seeded in six-well plates and transfected with 0.5 µg of psiRNA using Lipofectamine 2000 (Invitrogen). After 48 h the level of silencing was determined and cells were used for activation assays.

Confocal microscopy

Human primary cardiac cells on microchamber culture slides (Laboratory-tek, Gibco) were incubated with Alexa 488-CBV3 ssRNA (20 µg ml−1) for different time points, and were subsequently rinsed twice in PBS/0.02% BSA, before fixation with 4% formaldehyde for 15 min. The cells were fixed in order to prevent potential re-organization of the proteins during the course of the experiment. Cells were permeabilized using PBS/0.02% BSA/0.02% Saponin and labelled with EEA1 antibody for endosomes, and TLR7, TLR8 or MyD88 followed by incubation with the appropriate fluorescently labelled secondary antibody.

Cells were imaged on a Carl Zeiss LSM510 META confocal microscope (with an Axiovert 200 fluorescent microscope) using a 1.4 NA 63× Zeiss objective. The images were analysed using LSM 2.5 image analysis software (Carl Zeiss).

References

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. References
  • Akira, S. (2001) Toll-like receptors and innate immunity. Adv Immunol 78: 156.
  • Bieback, K., Lien, E., Klagge, I.M., Avota, E., Schneider-Schaulies, J., Duprex, W.P., et al. (2002) Hemagglutinin protein of wild-type measles virus activates toll-like receptor 2 signalling. J Virol 76: 36073613.
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