Findings

Dengue virus (DENV) is a member of the Flavivirus genus of the Flaviviridae family of enveloped, positive-strand RNA viruses. Four distinct serotypes (DENV1-4) of dengue viruses are transmitted to humans through the bites of the mosquito species, Aedes aegypti and A. albopictus. DENV causes a spectrum of disease in human, from acute febrile illness dengue fever (DF) to life-threatening dengue hemorrhagic fever/dengue shock syndrome (DHF/DSS). It has been estimated that about 50–100 million cases of DF, and about 250,000–500,000 cases of DHF occur worldwide every year. Furthermore, 2.5 billion of people are at risk for infection in subtropical and tropical regions of the world in the absence of effective intervention [1, 2]. It is hypothesized that immunological mechanisms play a key role in the pathogenesis of dengue infection. Although several gene expression profiling studies of dengue virus-infected cells have been carried out previously [3–6], little is currently known about the molecular mechanism of how Th genes and Th-related genes are activated and implicated in the immuno-pathogenesis of DENV infection.

In this study, we conducted a novel focused gene set, array-based platform of transcriptional Real Time (RT)-PCR analysis of human cell response to DENV infection during peak virus production and focused on genes which are related to the three classes of helper T cells. The three classes of 84 helper T cells (Th1-Th2-Th3) genes were simultaneously profiled using the RT-PCR array (Table 1). Comparative profiling of the Th genes was carried out on three different human cell types (monocytes, T-cells and hepatocytes). The data generated from the RT-PCR array profiling will enable us to determine the crucial virus-cell interactions that take place during DENV infection. The RT-PCR arrays revealed differential regulation of 41 Th genes among the different human cell types, these genes include cytokine genes representative of Th1, Th2 and Th3 cells, transcriptional factors that regulate the expression of these cytokines as well as other markers of CD4+ T lymphocytes. Genes involved in immune cell activation and the Th1 and Th2 type immune responses were also noted. From these, 20 genes were identified and these genes may contribute to the immuno-regulation of DENV pathogenesis. This study provided a first insight to the differential regulation of Th genes during DENV infection of different human cell types.

Table 1 Complete list of genes tested in the Th genes RT-PCR array.
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Dengue virus is known to be able to infect different kinds of cell types in human [7]. In this study, the susceptibility of human cell lines, K562, Jurkat and HepG2 to DENV 2 virus infection was first determined. K562 is a myelogenous cell line with monocyte and granulocyte properties [8]. Jurkat is a T cell lymphoblast-like cell line [9] and HepG2 is a heptocellular liver cell line [10]. Both K562 and Jurkat cells were selected for analysis as they are of immune origin and the HepG2 cell line was selected since dengue virus is hepatotrophic. The use of human cell lines for this study has been given approval by the National University of Singapore Institutional Review Board. These human cell lines were first subjected to low passage human isolate DENV (serotype 2, Singapore strain Den2ST) infection at a multiplicity of infection of 10. At different time points post-infection, virus containing supernatant were harvested for plaque assays and the DENV-infected cells were stained for immunofluorescence detection of viral antigen (envelope protein) production via flow cytometry. All the three human cell lines are shown to be highly susceptible to dengue virus infection, producing reasonably high virus titers by 3 days post-infection (Figure 1a). Production of viral antigen was also detected in all the three cell lines by 3 days post-infection (Figure 1b).

Figure 1
figure 1

Infectivity of human cell lines with DENV. (a) Growth curves of DENV in different human cell types. (b) Detection of DENV viral antigen (envelope protein) in different human cell types via flow cytometry analysis. All the human cells were shown to support DENV replication and high infectious virus titers were also obtained from the cells at 3 days post-infection.

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Next, all the three human cell lines were infected with DENV2 and processed for RT-PCR array experiments. Three independent DENV infection experiments were carried out to ensure the reproducibility of the changes in gene expression. Total RNA of the human cells (K562/Jurkat/HepG2) was extracted using the Qiagen RNeasy Kit. The quality and integrity of the total RNA extracted from mock-infected and dengue virus-infected cells on day 3 post-infection was first checked by running a portion of the total RNA on 1% agarose gel to detect for human ribosomal RNA, 28S and 18S. Human Th1-Th2-Th3 RT2 Profiler™ PCR Array (Superarray) was used for the simultaneous profiling of 84 genes related to the three classes of helper T cells. RT2 First Strand Kit (Superarray) was used for reverse transcription to obtain cDNA from total RNA. Five housekeeping genes (β-2-microglobulin, hypoxanthine phosphoribosyltransferase 1, ribosomal protein L13a, glyceraldehyde-3-phosphate dehydrogenase and β-actin) were included in the array to minimize functional biases.

Genes with fold value more than 2 in absolute value were considered to be differentially regulated. P-value of 3 independent control experiments and test experiments obtained for each gene was calculated. The differential regulation of the Th genes upon DENV infection of the human cell types are tabulated in Tables 2, 3 and 4. The Th genes are further classified based on their potential pathogenesis-induced classes (immune cell activation, inflammation, thrombocytopenia and vascular permeability) with reference to the description of the gene function summarized from Online Mendelian Inheritance in Man (OMIM), National Center for Biotechnology Information, National Library of Medicine http://www.ncbi.nlm.nih.gov/omim/ as of November 2008. From these genes, a group of 20 genes that may provide the molecular basis of the observed pathogenesis in the human cells upon DENV infection was identified (Tables 2, 3 and 4 – bolded). The criterion for the selection of these genes was based on their ability to enhance immune cell activation, inflammatory responses, thrombocytopenia as well as vascular permeability upon differential regulation. The complete gene expression profiles of all the three cell lines infected with DENV were provided (Figures 2, 3 and 4). The Th genes that were either up or down-regulated significantly in the different cell types were also indicated in the volcano plots as shown in Figures 2, 3 and 4. RANTES, CC-CKR-3, TLR6 and IL-6 were highly up-regulated in Jurkat and K562 cells upon infection with dengue virus. CD40L was shown to be significantly down-regulated in both Jurkat and HepG2 cells upon dengue virus infection.

Table 2 Differentially regulated Th genes of Jurkat cells infected with DENV.
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Table 3 Differentially regulated Th genes of K562 cells infected with DENV.
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Table 4 Differentially regulated Th genes of HepG2 hepatocytes infected with DENV.
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Figure 2
figure 2

Expression and statistical validation profiles of Th genes in dengue virus infected Jurkat cells. The differential regulation (up or down-regulated) of the 84 Th and Th-related genes upon dengue virus infection of Jurkat cells are shown. Genes with greater than 3Log2 fold increase are indicated in the respective graphs of each cell types. The volcano plot of the RT-PCR array for each of the cell types is also provided. The plot arranges Th genes along dimensions of differential regulation (either up or down-regulation – X axis) and statistical significance (Y-axis). The higher values on the Y-axis indicate statistical significant of the up or down-regulated genes.

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Figure 3
figure 3

Expression and statistical validation profiles of Th genes in dengue virus infected K562 cells. The differential regulation (up or down-regulated) of the 84 Th and Th-related genes upon dengue virus infection of K562 cells are shown. Genes with greater than 3 Log2fold increase are indicated in the respective graphs of each cell types. The volcano plot of the RT-PCR array for each of the cell types is also provided. The plot arranges Th genes along dimensions of differential regulation (either up or down-regulation – X axis) and statistical significance (Y-axis). The higher values on the Y-axis indicate statistical significant of the up or down-regulated genes.

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Figure 4
figure 4

Expression and statistical validation profiles of Th genes in dengue virus infected Hep G2 cells. The differential regulation (up or down-regulated) of the 84 Th and Th-related genes upon dengue virus infection of HepG2 cells are shown. Genes with greater than 3 Log2fold increase are indicated in the respective graphs of each cell types. The volcano plot of the RT-PCR array for each of the cell types is also provided. The plot arranges Th genes along dimensions of differential regulation (either up or down-regulation – X axis) and statistical significance (Y-axis). The higher values on the Y-axis indicate statistical significant of the up or down-regulated genes.

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Dengue virus infection causes dengue fever (DF), dengue hemorrhagic fever (DHF), and dengue shock syndrome (DSS), whose pathogenesis are not clearly understood. Current hypotheses of antibody-dependent enhancement, virus virulence, and IFN-gamma/TNF α-mediated immuno-pathogenesis are insufficient to explain clinical manifestations of DHF/DSS such as thrombocytopenia and hemoconcentration [11]. Furthermore, Chaturvedi and co-workers (1999) documented a shift from a Th1 response in mild dengue to a Th2 response in severe dengue haemorrhagic fever [12]. Increased serum levels of IL-4, IL-6 and IL-10 were observed mainly in cases of DHF grades III and IV. In contrast, the levels of IFN-γ and IL-2 were highest in cases of DF and low in DHF grade IV. Hence, these data indicated the importance of Th genes in mediating the pathogenesis of DENV infection.

In this study, we have been able to identify 20 Th and Th-related genes from the RT-PCR arrays that may contribute to the molecular basis of DENV pathogenesis. The 20 genes were selected based on their differential regulation and their implications in mediating immune cell activation, inflammatory responses, thrombocytopenia and vascular permeability during DENV infection. Nevertheless, the involvement of these genes on the molecular pathogenesis of DENV infection warrant further cohort studies on patients with DENV infection and these studies are currently being conducted in our laboratory.

Jurkat and K562 cells being immune cells have more differentially regulated Th related genes upon DENV infection when compared with HepG2 hepatocytes (Tables 2, 3 and 4). Fas ligand, IL-2 and IFN-γ up-regulation in Jurkat cells were also reported in studies using DENV-infected T cell clones [13]. Up-regulation of RANTES and IL-6 in dengue 2 virus-infected K562 cells was also reported by King and co-workers, showing elevated RANTES and IL-6 in DENV-infected mast cells and monocytes [14]. An earlier study by Liu and co-worker reported the expression of CD69 on monocytes at day 4 after the onset of fever which also substantiated the observation of CD69 up-regulation in DENV-infected K562 cells in this study [15]. Hence, the consistency of the data obtained from this study with previous published studies provided this experimental approach with confidence.

The observation of the few similar Th related gene regulation between the three cell lines suggested that the DENV triggered Th gene expression that was cell-type dependent in the RT-PCR array. Another gene profiling study performed on DENV-infected cells also observed cell-type specific gene changes [6]. Nevertheless, a group of Th1 and Th2 genes inducing cell activation and inflammation (GMCSF, RANTES, TLR6 and CC-CKR-3) were found to be significantly up-regulated in both K562 and Jurkat cells, this may imply the presence of potential cross-talking pathways that mediate common inflammatory responses during DENV infection. More studies are now being conducted to further verify this interesting observation. The over-expression or inappropriate inflammation can lead to tissue destruction commonly observed in DSS/DHF [16].

For dengue virus infection, the severity of the disease is best indicated by the extent of plasma leakage. A major group of genes (GMCSF, RANTES, IL2, INFγ, TLR6, CCR2A, IL-6, CKR-4) causing thrombocytopenia and vascular permeability was also found to be up-regulated in all three cell types. These gene products are known mediators of thrombocytopenia and vascular permeability. In consistent with previous studies, some of these genes have also been reported to be up-regulated during dengue infection, including RANTES [17] and IL-6 [18]. In addition, the down-regulation of IL-4 was observed in K562 cells and this has important implication in causing vascular permeability [19]. The over-expression of these cellular Th genes might play a crucial role in enhanced production of anti-platelet or anti-endothelial cell autoantibodies, elevated levels of tPA, as well as a deficiency in coagulation. Therefore, damaged endothelial cells may upset the procoagulant/anticoagulant balance of endothelium and increase the tendency of bleeding. Activated endothelial cells may also contribute to the development of thrombocytopenia by sequestering platelets [20].

In summary, the focused Th genes RT-PCR array profiling showed a complex network of DENV-induced cell interactions during infection. The array-based Th gene profiles documented in this study may also serve as potential biomarkers for DENV infection. Nevertheless, further verification of these differentially regulated Th genes on DENV-infected patient samples will be helpful. Comparative profiling of Th genes in different human cell types in response to DENV infection may therefore provide interesting insights into the possible molecular mechanisms behind the observed cyto-pathology in DENV infection.