Dengue virus induces thrombomodulin expression in human endothelial cells and monocytes in vitro

doi:10.1016/j.jinf.2009.02.018

Journal of Infection

Volume 58, Issue 5, May 2009, Pages 368-374

https://doi.org/10.1016/j.jinf.2009.02.018 Get rights and content

Summary

Objectives

Dengue virus (DV) infections can cause severe life-threatening dengue hemorrhagic fever/dengue shock syndrome (DHF/DSS). However, the mechanism to cause hemorrhage in DV infections remains poorly understood. Thrombomodulin (TM), expressed on the surface of endothelial cells and monocytes, is very important in regulation of coagulation and inflammation. Therefore, the effect of DV on the TM expression was studied in vitro using both endothelial cells and monocytes.

Methods and results

The expression of TM in human endothelial cell line, HMEC-1, monocytic cell line THP-1 and peripheral blood mononuclear cells derived from human blood was increased after DV infection, UV-inactivated DV or recombinant DV envelop protein domain III stimulation as demonstrated by flow cytometry and immunofluorescent staining. Western blot analysis further confirmed only DV but not enterovirus 71 infection of HMEC-1 cells increased TM protein expression. In addition, RT-PCR analysis showed the increase of TM mRNA as well as other protein C activation-related molecules in DV stimulated HMEC-1 in a dose-dependent manner.

Conclusion

These results suggest that DV stimulation of human endothelial cells and monocytes can increase the expression of TM, which may contribute to the anticoagulant properties of cells during DV infection.

Introduction

Dengue virus (DV) infection which is transmitted by mosquitoes Aedes aegypti and Aedes albopictus is prevalent in over 100 countries especially in tropical and subtropical areas and threatens the health and life of more than 2.5 billion people.1, 2 According to disease severity, DV infection may generally, result in mild dengue fever (DF) or severe life-threatening dengue hemorrhagic fever/dengue shock syndrome (DHF/DSS).3, 4, 5 Most DF patients will be spontaneously recovered. However, in some cases, especially during secondary infection with different serotypes of DV, DHF/DSS may develop. Even though antibody dependent enhancement (ADE) has been proposed to explain why DHF/DSS occurs mostly in secondary infection with different serotype of DV,⁶ the mechanism of hemorrhage induced by DV infection is not yet fully understood.

Thrombomodulin (TM) is a 75 kDa transmembrane glycoprotein expressed mainly on the surface of vascular endothelium. However, TM has also been detected in human monocytes and a variety of other tissues.7, 8 TM acts as a vascular endothelial cell receptor for thrombin to activate protein C (PC).⁹ Activation of PC by the thrombin-TM complex is further enhanced when PC is bound to the endothelial cell protein C receptor (EPCR).9, 10 Activated PC dissociates from EPCR and binds to protein S on appropriate cell surfaces where it inactivates factors Va and VIIIa, and thereby inhibits further thrombin generation. Therefore, TM plays an important role in the anti-coagulant state of endothelium. On the other hand, circulating high levels of soluble TM may be associated with ongoing endothelial damage or ongoing inflammation during disease course.¹¹ An elevated serum level of soluble TM has been reported in dengue patients.12, 13, 14 In addition, DHF/DSS patients have higher concentrations of soluble TM than those with DF.12, 13, 14 However, it is unclear whether the increased soluble TM in dengue patients is due to the increase of TM expression in DV-infected endothelial cells or due to the increase of endothelial damage in dengue patients. In this study we tried to explore this question by using live and UV-inactivated DV (UV-DV) to stimulate human microvascular endothelial cell line (HMEC-1) and human acute monocytic cell line (THP-1) in vitro to understand the effect of DV on the expression of TM in these cells. The results indicated that DV antigen stimulation can increase TM expression on HMEC-1 and THP-1, which may contribute to the anti-coagulant state of these cells during DV infection.

Section snippets

Preparation of virus stock and virus titration

Dengue virus serotype 2 (strain PL0046) was propagated in C6/36 cells. Briefly, monolayers of C6/36 were inoculated with the virus at multiplicity of infection (MOI) of 0.1 and incubated at 26 °C, 5% CO₂ for 5 days. The culture medium was harvested and cell debris was removed by centrifugation at 900 × g for 10 min. After further centrifugation at 16,000 × g for 10 min, the virus supernatant was collected and stored at −80 °C until use. Virus titer was determined by plaque assay using BHK-21 cell line.

Immunofluorescent staining and flow cytometric assay

After infection for 24 h, cells were fixed with 4% paraformaldehyde for 30 min, then permeabilized with 0.5% Triton X-100 for 10 min. Cells were then washed with PBS and blocked with 0.05% BSA in PBS. Fixed cells were stained with primary antibody at 4 °C for 1 h. Mouse anti-human TM antibody (Santa Cruz Biotechnology, Santa Cruz, CA) or rabbit anti-human TM antibody (Santa Cruz Biotechnology) was used as primary antibodies. After being washed, the cells were incubated with secondary antibody.

Reverse-transcription polymerase chain reaction (RT-PCR)

RNA was extracted by using an isolation Reagent (Trizole; Life Technologies Inc., Rockville, MD) and quantified at 260 nm. Reverse-transcription (RT) was performed by using a kit (Gibco) according to the manufacturer's instructions. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) served as a control. The primers used to amplify GADPH, EPCR, protein S, tissue plasminogen activator (t-PA) and TM were listed in Table 1. A total reaction volume of 20 μl contained 4 μl of RT product, 2.5 units of Taq

Statistical analysis

Data are expressed as mean ± SD. Student's t test was used to analyze the significance of the difference between the test and the control groups. Statistical significance was set at P < 0.05.

Both live and UV-DV increased TM expression in HMEC-1 cells

DV infection of HMEC-1 increased TM expression in a dose-dependent manner as demonstrated by indirect immunofluorescent staining (Fig. 1). In addition, cell morphology was changed from round shape to irregularly protruding of the cytoplasm in DV-infected HMEC-1 at MOI = 10, which may be due to the cytopathic effect induced by DV infection at high MOI.¹⁷ UV-DV stimulation also increased TM expression in HMEC-1 cells. However, unlike DV infection-induced TM expression which was more homogeneously

Discussion

Systemic inflammation induced during infection generally shifts the hemostatic mechanisms in favor of thrombosis.21, 22 However, infection with DV and certain viruses can tilt the hemostasis toward bleeding and cause viral hemorrhage fever (VHF).²³ Advances in our understanding of the mechanism to cause hemorrhage by DV may, therefore, lead to the recognition of potentially useful treatment against VHF.

Both endothelial cells and monocytes play important roles in regulating hemostasis and both

Acknowledgements

This work was supported by the grant NSC96-2628-B006-006-MY3 from the National Science Council, Taiwan.

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Dengue virus induces thrombomodulin expression in human endothelial cells and monocytes in vitro

Summary

Objectives

Methods and results

Conclusion

Introduction

Section snippets

Preparation of virus stock and virus titration

Immunofluorescent staining and flow cytometric assay

Reverse-transcription polymerase chain reaction (RT-PCR)

Statistical analysis

Both live and UV-DV increased TM expression in HMEC-1 cells

Discussion

Acknowledgements

J Infect Chemother

Lancet Infect Dis

Blood

Chest

Vaccine

J Infect

Maturitas

J Thromb Haemost

Lancet Infect Dis

J Biol Chem

J Biol Chem

FEMS Immunol Med Microbiol

Global spread and persistence of dengue

Annu Rev Microbiol

The dengue viruses

Clin Microbiol Rev

Dengue and dengue hemorrhagic fever

Clin Microbiol Rev

Antibody, macrophages, dengue virus infection, shock, and hemorrhage: a pathogenetic cascade

Rev Infect Dis

Thrombomodulin: an overview and potential implications in vascular disorders

Lupus

The endothelial cell protein C receptor augments protein C activation by the thrombin-thrombomodulin complex

Proc Natl Acad Sci U S A