Introduction
EV71 is a member of the
Picornaviridae family, which is composed of a large number of small non-enveloped, positive strand RNA viruses with a genome size of approximately 7500 bp [
1],[
2]. Increasing evidences indicate that EV71 has become the major etiological agent of current outbreaks of hand, foot, and mouth disease (HFMD) in the Asia-Pacific region, including China [
3]-[
5]. In addition, the clinical features of EV71 infection vary from mild HFMD or herpangina to aseptic meningitis, encephalitis, pulmonary edema and even death [
6]-[
8]. Premature or impaired immunity upon EV71 infection has been associated with increased morbidity and mortality [
9],[
10]. However, the molecular pathogenesis of EV71 infection remains elusive.
Mitogen activated protein kinases (MAPKs) are central molecules mediating signaling pathways in innate immunity. They belong to a family of serine/threonine protein kinases which are largely conserved among eukaryotes and involved in many cellular processes such as inflammation, proliferation, differentiation, movement, and death [
11],[
12]. To date, seven distinct groups of MAPKs have been characterized in mammalian cells, including extracellular regulated kinases (ERK1/2), c-Jun N-terminal kinases (JNK1/2/3), p38 MAPK (p38 α/β/γ/δ), ERK3/4, ERK5, ERK7/8 and Nemo-like kinase (NLK) [
13],[
14]. Among these, the most extensively studied are ERK1/2, JNKs and p38 MAPKs [
15],[
16]. Acute activation of MAPK signaling cascades is utilized by both viruses for their replication and hosts for defense against the viruses [
17],[
18]. Virus infection activates the MEK/ERK signaling pathway, which further promotes activation of transcription factors and increases the secretion of different cytokines to affect virus propagation [
19],[
20]. Furthermore, transcription of many MAPK-regulated pro-inflammatory or antiviral cytokines such as IL-2, IL-6, IFN-β and TNF-α is dependent on transcription factor NF-κB, c-Fos and c-Jun.
DCs are the first line of host defense. They initiate specific host immune responses by capturing, processing, and presenting antigens through MHC-I and MHC-II molecules on the cellular surface and activating naïve T cells [
21],[
22]. The effect of MEK/ERK signaling cascades during EV71 infection in many other cells has been extensively explored. For example, it has been reported that the MEK/ERK signal cascade is required for replication of EV71 in embryonic rhabdomyosarcoma (RD) cells and HEK cells [
23],[
24]. However, MEK/ERK signaling pathway in EV71-infected iDCs remains largely unknown.
To explore the roles of MEK/ERK signaling pathway in EV71-infected iDCs, we induced iDCs from PBMCs in the presence of granulocyte-macrophage colony-stimulating factor (GM-CSF) and IL-4. The iDCs were then infected with EV71 and used to investigate the effect and mechanism of MEK/ERK signaling pathway associated molecules during EV71 infection.
Discussion
DCs are essential for the induction of specific immune responses against invading pathogens [
17],[
25]. Two distinct DC subsets were originally defined in human blood based on the expression of CD11c. The CD11c
+ DCs are generally called myeloid DCs (mDCs) because they express myeloid markers, whereas CD11c
− DCs are called plasmacytoid DCs (pDCs) because of their plasma cell-like morphology and localization at the precursor stage [
26],[
27]. Human DCs, especially iDCs, are highly specialized and efficient in uptaking and processing antigens, which have been shown to support the propagation of several viruses including EV71 and dengue viruses, and the infection of these viruses could increase the viability, activation, cytokine release and T-cell priming activity in DCs [
28],[
29].
As previously reported, herpes simplex virus could infect both mature and immature DCs, whereas only iDCs support a productive infection, which could lead to apoptosis [
30],[
31]. EV71 could also infect both mature and immature DCs, and replicate in them even though at slightly less and comparable amounts [
28]. In this study, we isolated mononuclear cells from peripheral blood, purified monocytes and differentiated them into iDCs in the presence of GM-CSF plus IL-4. Subsequently, iDCs were infected by EV71 with a MOI of 5. Previous report showed that EV71 could activate iDCs and promote their T-cell priming activity, but failed to induce the maturation (CD83 expression) of iDCs [
14]. However, we found that EV71 infection increased the percentages of CD83 and CD86 were expressed on iDCs. It might be related to viral titer, infection duration and experiment performance, and needed to be further studied. In addition, the percentages of CD80 and HLA-DR were not changed in EV71-infected iDCs. These results showed that EV71 infection could enhance the expression of maturation markers of iDCs and activate iDCs.
There are mainly three MAPK signaling pathways associated with virus infection, including ERK1/2, p38 MAPK, and JNK [
32]-[
35]. In contrast to the JNK cascade, activation of the Raf/MEK/ERK pathway in host cells (e.g., RD cells, HEK293 cells, vero cells, oval cells, and lymphocytes) seems to be required for virus replication, including EV71, herpes simplex virus 2, yellow fever virus, hepatitis B virus and human immunodeficiency virus type 1 [
23],[
24],[
36]-[
38]. Here, we showed that the gene expression levels of KRAS, Raf-1, c-Fos, c-Jun, c-myc, and Elk1, but not MEK1/2 and ERK1/2 in EV71-infected iDCs increased at 1/2 h, 2 h, 8 h and 24 h p.i., suggesting that EV71 infection could activate Ras and Raf-1, which in turn enhanced activation and phosphorylation of MEK1/2 and ERK1/2. Phosphorylated ERK1/2 subsequently translocated into nucleus, where they upregulated the transcription of c-Fos and c-Jun to promote EV71 replication. Furthermore, we found that the treatment with U0126 (20 μM) significantly reduced phosphorylation of MEK1/2 and ERK1/2, as well as their downstream molecules c-Fos, c-Jun, c-myc and Elk-1. Because the activation of these transcription factors participated in the regulation of cell proliferation and differentiation, we hypothesize that the activation of MEK/ERK signal cascade is required for EV71 replication in iDCs.
SOS was discovered in Drosophila melanogaster and plays an important role in normal eye development in Drosophila [
39],[
40]. Human SOS (hSOS) has two homologues, SOS1 and SOS2, which are ubiquitously expressed in different human tissues and cell lines [
41]. Upon growth factor stimulation, SOS1 is recruited by growth factor receptor-bound protein 2 (Grb2) to the plasma membrane with subsequent release of autoinhibition. Once SOS1 is activated, it interacts with Ras proteins to promote guanine nucleotide exchange (GDP/GTP) and subsequent formation of the active Ras-GTP complex which stimulates the activation of Ras/MEK/ERK sigaling pathway [
42],[
43]. In this study, we found that EV71 infection significantly upregulated the expressions of SOS1 mRNA level and protein, which might further enhance the activation of Ras/MEK/ERK signaling pathway. Although the mechanism of SOS1 overexpression stimulated by EV71 infection is still unknown yet, we speculate that SOS1 plays an important role in EV71 infected-iDCs.
Several pro-inflammatory cytokines (e.g. IL-1, IL-6, TNF-α, and IFN-β, etc) are induced by oxidant stress, cytokines, and virus infection, which play a role in host cell damage, chronic inflammation, and other immunoresponses [
44],[
45]. EV71 infection can induce iDCs to secrete various cytokines. PCR array showed that EV71 infection increased the expression of IL-1α, IL-2, IL-6, IL-12, TNF-α, IFN-β and IFN-γ, but not IFN-α1, by 2.04-5.49-fold in iDCs. In addition, EV71 infection stimulated the secretion of IL-1α, IL-2, IL-6, IL-12, TNF-α,IFN-β and IFN-γ as measured by luminex fluorescent technique. For these cytokines, a good correlation was obtained between PCR array and luminex data. Furthermore, treatment with ERK1/2 inhibitor U0126 (20 μM) decreased the production of IL-1α, IL-2, IL-6 and TNF-α through blocking the MEK/ERK signaling pathway.
Materials and methods
Ethics statement
The study was approved by the Ethics Committee of the Third Affiliated Hospital of Soochow University, and all patients provided informed consents.
Antibodies and chemicals
Dulbecco's modified Eagle's medium (DMEM), fetal bovine serum (FBS) and RPMI 1640 were purchased from Thermo Scientific HyClone (UT, USA). Hybond C membrane and ECL Western blot detection system were from Pierce (Rockford, IL, USA). Rabbit polyclonal antibodies against MEK1/2, p-MEK1/2, ERK1/2, p-ERK1/2, c-Fos, p-c-Fos, c-Jun, p-c-Jun, p-c-myc, c-myc, Elk1, p-Elk1, and HRP conjugated goat anti-rabbit IgG were purchased from SAB (Pearland, TX, USA). SOS1 mouse monoclonal antibody was provided from abnova Company (Taiwan). Antibodies against anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were obtained from ProteinTECH Group (Chicago, IL, USA). Rabbit polyclonal antibody against EV71/VP1 was purchased from Abcam Company (Cambridge, UK). The MEK1/2 specific inhibitor U0126 was acquired from LC Laboratories (Woburn, MA, USA) and freshly prepared as DMSO solution.
Cell culture and virus propagation
RD cells were cultured in high glucose DMEM (HyClone, USA) supplemented with 10% FBS at 37°C and in a humidified incubator with 5% CO
2 atmosphere. When cells reached 90% confluence, the original media were removed and the monolayer cells were washed with PBS once. Approximately 1 × 10
6 of RD cells were incubated with EV71 strain GDV083 (ATCC VR-784) at a MOI of 5 or as indicated and allowed for absorption for 1.5 h at 37°C. Infected cells and culture supernatants were collected at different time intervals. Viruses were propagated in up to 90% confluent cell monolayer in DMEM containing 2% FBS and antibiotics as described above. The viral titer was determined by cytopathic effects (CPE) and expressed as 50% tissue culture infective dose (TCID50) per ml [
46].
Generation of iDCs
PBMCs were isolated from healthy blood donors and purified by Ficoll-Hypaque (Invitrogen, CA, USA) density gradient centrifugation. Monocytes were isolated from PBMCs by adhesion to plastic dishes for more than 2 h at 37°C and subsequently cultured with GM-CSF (100 ng/ml) and IL-4 (50 ng/ml). iDCs were generated for 7 days and infected with EV71 at a MOI of 5 for 1 h at 37°C. After washed twice with PBS, iDCs were cultured in RPMI medium for 24 h, and unifected iDCs were used as control. The phenotypic patterns of CD3, CD11c, CD80, CD83, CD86 and HLA-DR were characterized by flow cytometry (Beckman coulter, CA, USA). Meanwhile, the supernatants were stored at -80°C.
Total RNA preparation and PCR arrays
After incubating at 37°C for 1/2 h, 2 h, 8 h and 24 h, total cellular RNA was isolated from EV71-uninfected and infected iDCs using the Trizol reagent. Complementary DNA (cDNA) was generated from mRNA by reverse transcription with oligo (dT) primer. Then, 1 μg of cDNA was added into a 96-well plate pre-dispensed with the indicated primers (CT biosciences, China). PCR arrays were performed by LightCycler 480 (Roche Diagnostics, Germany). The thermocycler parameters were performed with an initial denaturation at 95°C for 5 min followed by 40 cycles of denaturation at 95°C for 15 s, annealing at 60°C for 15 s and extension at 72°C for 20 s. The housekeeping genes such as B2M, ACTB, GAPDH, RPL27, HPRT1 and OAZ1 were used to normalize RNA amount. Fold changes were calculated using the formula of 2-ΔΔCt. Each experiment was performed in triplicate.
Cell extraction and western blot analysis
iDCs were pre-incubated for 1 h with U0126 at different concentrations as indicated and then infected with EV71 at a MOI of 5 in the presence of U0126 for 48 h. Cells were harvested by centrifugation, washed and lysed with 50 mM Tris pH 6.8, containing 250 mM NaCl, 2% sodium deoxycholate, 0.5% NP-40 and 1 mM PMSF. After vortexing and incubating on ice for 10 min, cell lysates were centrifuged at 10,000 × g for 5 min. Total protein concentration was determined by the bicinchoninic acid protein assay kit (Pierce). Proteins in the lysates were separated by sodium dodecyl sulfate polyacrylamide-polyacrylamide gel electrophoresis (SDS-PAGE). The separated proteins were transferred to PVDF membranes (Millipore), and probed with specific primary antibodies. After washed with PBS, the membranes were incubated with horseradish peroxidase conjugated secondary antibodies. The specific proteins were detected by ECL reagents (GE Healthcare), visualized on Super RX film (Fujifilm) and quantitated by densitometric analysis (ImageQuant, Molecular Dynamics and PDSI, GE Healthcare). The relative amounts of the phospho-ERK1/2 (p-ERK1/2) were normalized to GAPDH after densitometry scanning and analysis with the band leader software (version 3.0).
Evaluation of cytokine levels by luminex fluorescent technique
iDCs were infected with EV71 at a MOI of 5 for 1 h at 37°C, washed twice and cultured in complete RPMI medium. At 8 h, 12 h, and 24 h p.i., the supernatants were collected by centrifugation (2,000 × g). As previously reported [
47], the concentrations of IL-1α, IL-2, IL-6, IL-12, TNF-α, IFN-α1, IFN-β and IFN-γ in culture supernatant were analyzed with Milliplex Magnetic Beads (Millipore, USA) using luminex fluorescent technique.
Statistic analysis
Quantitative data were presented as the mean ± SE and statistically analyzed using GraphPad Prism software (San Diego, CA). Qualitative data were analyzed with Chi-square test. A p value less than 0.05 was considered as being statistically significant.
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Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
WS and JJ conceived and designed the experiments, participated in the data analysis and manuscript preparation. XH, HP and LZ performed cell culture, agar gel electrophoresis, Western blot and flow cytometry. YL and ZG participated in the data analysis and manuscript preparation. QJ, MS and YJ performed PCR-fluorescence probing assay, agar gel electrophoresis and analyzed the data. All authors have read and approved the final manuscript.