PLOS Neglected Tropical Diseases Dengue Virus Activates Membrane TRAIL Relocalization and IFN-α Production by Human Plasmacytoid Dendritic Cells in vitro and in vivo --Manuscript Draft-Manuscript Number:
PNTD-D-12-01360R1
Full Title:
Dengue Virus Activates Membrane TRAIL Relocalization and IFN-α Production by Human Plasmacytoid Dendritic Cells in vitro and in vivo
Short Title:
Dengue Virus Activates Plasmacytoid Dendritic Cell
Article Type:
Research Article
Keywords:
dengue virus; plasmacytoid dendritic cells; TRAIL; interferon; microscopy
Corresponding Author:
Jean-Philippe Herbeuval, PhD CNRS Paris, FRANCE
Corresponding Author Secondary Information: Corresponding Author's Institution:
CNRS
Corresponding Author's Secondary Institution: First Author:
Mariana Gandini
First Author Secondary Information: Order of Authors:
Mariana Gandini Christophe Gras Elzinandes Leal Azeredo Luzia Maria de Oliveira Pinto Nikaïa Smith Philippe Despres Rivaldo Venâncio da Cunha José Luiz de Souza Claire Fernandes Kubelka Jean-Philippe Herbeuval, PhD
Order of Authors Secondary Information: Abstract:
Background Dengue displays a broad spectrum of clinical manifestations that may vary from asymptomatic to severe and even fatal features. Plasma leakage/ hemorrhages can be caused by a cytokine storm induced by monocytes and dendritic cells during dengue virus (DENV) replication. Plasmacytoid dendritic cells (pDCs) are innate immune cells and in response to virus exposure secrete IFN-α and express membrane TRAIL (mTRAIL). We aimed to characterize pDC activation in dengue patients and their function under DENV-2 stimulation in vitro. Methods & Findings Flow cytometry analysis (FCA) revealed that pDCs of mild dengue patients exhibit significantly higher frequencies of mTRAIL compared to severe cases or healthy controls. Plasma levels of IFN-α and soluble TRAIL are increased in mild compared to severe dengue patients, positively correlating with pDC activation. FCA experiments showed that in vitro exposure to DENV-2 induced mTRAIL expression on pDC. Furthermore, three dimension microscopy highlighted that TRAIL was relocalized from intracellular compartment to plasma membrane. Chloroquine treatment inhibited DENV-2-induced mTRAIL relocalization and IFN-α production by pDC. Endosomal viral Powered by Editorial Manager® and Preprint Manager® from Aries Systems Corporation
degradation blockade by chloroquine allowed viral antigens detection inside pDCs. All those data are in favor of endocytosis pathway activation by DENV-2 in pDC. Coculture of pDC/DENV-2-infected monocytes revealed a dramatic decrease of antigen detection by FCA. This viral antigens reduction in monocytes was also observed after exogenous IFN-α treatment. Thus, pDC effect on viral load reduction was mainly dependent on IFN-α production Conclusions This investigation characterizes, during DENV-2 infection, activation of pDCs in vivo and their antiviral role in vitro. Thus, we propose TRAIL-expressing pDCs may have an important role in the outcome of disease. Suggested Reviewers:
Eva Harris Center for Global Public Health
[email protected] Michael Diamond Washington University of St Louis
[email protected] Esper Kallas University of São Paulo
[email protected] Marie-Lise Gougeon Institut Pasteur
[email protected] MLG is an expert in host-virus interactions.
Opposed Reviewers:
Maria Guzman Center for Viral Diseases, Pedro Kour Tropical Medicine Institute, Ciudad Habana, Cuba Competitive interests Allan Rothman University of Rhode Island Competitive interest Irene Boch Mt. Sinai University Competitive interest
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Original - Cover Letter
Dear Editors, Please find bellow an abstract of the manuscript entitled “Dengue virus activates membrane TRAIL relocalization and IFN-α production by human plasmacytoid dendritic cells in vitro and in vivo ”, by Gandini et al. to be considerate for publication in Plos Neglected Tropical Diseases. Dengue fever (DF) displays a broad spectrum of clinical manifestations that may vary from asymptomatic to severe and even fatal features. Although the immune pathogenesis is not completely understood, plasma leakage/ hemorrhages could be caused by a cytokine storm induced by monocytes and dendritic cells during Dengue virus (DENV) replication. Plasmacytoid dendritic cells (pDCs) bridge innate and adaptive immunity and have a central role in defense against viruses. These cells can be activated in response to virus exposure secreting IFN-α and expressing plasma membrane TRAIL (mTRAIL) (Colisson et al, Blood, 2010). However, a role for activated pDCs during DF is still unclear. We aimed to characterize pDC activation in vivo in DF patients and in vitro by DENV-2 stimulation. PDC function during infection of monocyte was evaluated too. We observed by flow cytometry that DF patients’ pDCs (n=30) exhibit more mTRAIL positive pDCs compared to severe DF (n=10) cases or healthy controls (n=20). Furthermore, levels of IFN-α and soluble TRAIL are increased in DF compared to Severe DF, correlating positively with pDC activation. Thus the more activated pDCs were the less severe disease was. Direct pDC activation by DENV-2 was confirmed by TRAIL relocalization from intracellular compartment to plasma membrane using flow cytometry and 3D microscopy. We demonstrate here that endocytosis pathway is crucial for pDC activation by DENV. Finally, IFN-α treatment could reduce viral antigen detection in infected monocytes (80% less than without IFN-α treatment). Using a co-culture approach, we observed that pDCs diminished DENV positive monocyte detection and that effect was mainly due to IFN-α production. This study therefore characterizes pDCs as potential antiviral cells for DENV-2. The novelty of our approach and results can be summarized by the following points: 1. This is the first study to assess activation profile of pDCs from a cohort of DF and sever Dengue fever patients. 2. We use for the first time 3D microscopy to detect viral antigens and TRAIL re-localization to pDCs membrane. 3. We show here a direct antiviral role for IFN-α in DENV-infected monocytes. 4. Finally, we demonstrated that pDCs cocultured with monocytes had strong antiviral function on DENV infection.
We believe that our study is related to the scope of this journal once Dengue is a Neglected Tropical Disease. We are contributing with new insights about the mechanisms of immune pathogenesis, especially about antiviral pathway to which dengue virus is susceptible. Finally, we suggest pDC activation as a good prognostic marker for therapeutic approaches and vaccines. Thank you for taking into consideration our manuscript for publication in your journal.
Sincerely yours,
Dr Jean-Philippe Herbeuval
Manuscript Click here to download Manuscript: Manuscript-revised.doc
1
Dengue Virus Activates Membrane TRAIL Relocalization and IFN-α Production
2
by Human Plasmacytoid Dendritic Cells in vitro and in vivo
3
Short Title: Dengue Virus Activates Plasmacytoid Dendritic Cell
4
Mariana Gandini1; Christophe Gras2; Elzinandes Leal Azeredo1; Luzia Maria de
5
Oliveira Pinto1;Nikaïa Smith2, Philippe Despres6, Rivaldo Venâncio da Cunha4; Luiz
6
José de Souza5; Claire Fernandes Kubelka1#; Jean-Philippe Herbeuval23#*
7 8 9
(1) Laboratório de Imunologia Viral, Instituto Oswaldo Cruz, FIOCRUZ; Rio de Janeiro, RJ, Brazil; (2) CNRS UMR 8147; Université Paris Descartes, Paris, France;
10
(3) CBNIT, CNRS UMR 8601 Université Paris Descartes, Paris, France
11
(4) Departamento de Clínica Medica, FM, Universidade Federal do Mato Grosso
12
do Sul, Campo Grande, MS, Brazil;
13
(5) Centro de Referencia em Dengue, Campos de Goytacases, RJ, Brazil.
14
(6) Unité des Interactions moléculaires Flavivirus-Hôtes, Institut Pasteur, Paris,
15 16 17
France (#) These authors contributed equally to this work. *Corresponding author:
[email protected]
18 19
1
1
Abstract
2
Background
3
Dengue displays a broad spectrum of clinical manifestations that may vary from
4
asymptomatic to severe and even fatal features. Plasma leakage/ hemorrhages can be
5
caused by a cytokine storm induced by monocytes and dendritic cells during dengue
6
virus (DENV) replication. Plasmacytoid dendritic cells (pDCs) are innate immune cells
7
and in response to virus exposure secrete IFN-α and express membrane TRAIL
8
(mTRAIL). We aimed to characterize pDC activation in dengue patients and their
9
function under DENV-2 stimulation in vitro.
10
Methods & Findings
11
Flow cytometry analysis (FCA) revealed that pDCs of mild dengue patients
12
exhibit significantly higher frequencies of mTRAIL compared to severe cases or healthy
13
controls. Plasma levels of IFN-α and soluble TRAIL are increased in mild compared to
14
severe dengue patients, positively correlating with pDC activation. FCA experiments
15
showed that in vitro exposure to DENV-2 induced mTRAIL expression on pDC.
16
Furthermore, three dimension microscopy highlighted that TRAIL was relocalized from
17
intracellular compartment to plasma membrane. Chloroquine treatment inhibited
18
DENV-2-induced mTRAIL relocalization and IFN-α production by pDC. Endosomal
19
viral degradation blockade by chloroquine allowed viral antigens detection inside pDCs.
20
All those data are in favor of endocytosis pathway activation by DENV-2 in pDC.
21
Coculture of pDC/DENV-2-infected monocytes revealed a dramatic decrease of antigen
22
detection by FCA. This viral antigens reduction in monocytes was also observed after
23
exogenous IFN-α treatment. Thus, pDC effect on viral load reduction was mainly
24
dependent on IFN-α production
25
Conclusions
26
This investigation characterizes, during DENV-2 infection, activation of pDCs in
27
vivo and their antiviral role in vitro. Thus, we propose TRAIL-expressing pDCs may
28
have an important role in the outcome of disease.
29 2
1
Author Summary
2
Dengue is an important endemic tropical disease to which there are no specific
3
therapeutics or approved vaccines. Currently several aspects of pathophysiology remain
4
incompletely understood. A crucial cellular population for viral infections, the
5
plasmacytoid dendritic cells (pDCs) was analyzed in this study. The authors found an in
6
vivo association between the activation state of pDCs and the disease outcome.
7
Membrane TNF-related apoptosis inducing ligand (TRAIL) expressing pDCs,
8
representing activated pDCs, were found in higher frequency in milder cases of dengue
9
than severe cases or healthy individuals. Detection of antiviral cytokine interferon-alpha
10
(IFN-α) and soluble TRAIL positively correlated with pDC activation. Dengue virus
11
(DENV) serotype-2 was able to directly activate pDCs in vitro. Under DENV
12
stimulation TRAIL was relocalized from intracellular to pDC plasma membrane and
13
IFN-α was highly produced. The authors suggest an endocytosis-dependent pathway for
14
DENV-induced pDC activation. It is also highlighted here a role for exogenous IFN-α
15
and pDCs in reducing viral replication in monocytes, one of DENV main target cells.
16
These findings may contribute in the future to the establishment of good prognostic
17
immune responses together with clinical manifestations/warning signs.
18
3
1
Introduction
2
Dengue is the most important arthropod-borne emerging viral disease in tropical
3
countries due to its high morbidity and risk of mortality [1]. For example, in Brazil,
4
dengue is a major public health problem and about two million cases were reported
5
during 2010-2012 [2]. Dengue virus (DENV) is a single-stranded RNA virus belonging
6
to genus Flavivirus [3,4]. All DENV serotypes (DENV-1 to -4) may induce a broad
7
spectrum of clinical manifestations from asymptomatic to severe clinical features,
8
characterized by hemorrhagic manifestations and a shock syndrome [5,6,7]. High viral
9
load may cause an exacerbated cytokine production that plays a key role in the
10
generation
11
monocytes/macrophages and dendritic cells are susceptible to viral replication
12
[10,11,12,13] and can release soluble mediators involved in vascular permeability and
13
plasma leakage besides coagulation disorders [14,15,16,17].
of
important
physiopathological
processes
[8,9].
Human
14
Dendritic cells link innate and adaptive immunity and play a key role in shaping
15
effective immune responses. Two major subpopulations are described: myeloid or
16
conventional dendritic cells (cDCs) and plasmacytoid dendritic cells (pDCs)
17
[18,19,20,21]. In contrast to cDCs, pDC are not found in homeostatic tissues but mainly
18
in circulating blood and in lymphoid tissues [21,22,23]. Despite being rare cells, pDCs
19
produce up to 1,000-fold more IFN-α than other cell types in response to virus exposure
20
[24]. Viral activation of pDCs can be regulated by either one of the two Toll-like
21
receptors (TLR), TLR-7 or TLR-9 [25], which are considered to be the pattern
22
recognition receptors (PRR) for RNA [26] and DNA [27], respectively. It has been
23
shown that cDC are efficiently infected by DENV and that viral replication blocked
24
cDC maturation [28,29]. However, unlike cDCs, it has been reported that pDCs are not
25
supporting productive DENV replication [30]. Indeed, DENV can activate pDCs
26
through cell endosomal activity and TLR-7 pathway [31]. Furthermore, dengue-infected
27
patients had impaired pDC activation features. Indeed, absolute numbers of blood pDC
28
were decreased [32,33] and low levels of serum IFN-α [34] were reported.
29
TNF-related apoptosis-inducing ligand (TRAIL) is a pro-apoptotic molecule,
30
which induces death of cells that express its death receptors (DR), DR4 and DR5
31
[35,36]. Furthermore, IFN-α regulates TRAIL expression by several cell types [37]. 4
1
Soluble or membrane TRAIL mediates apoptosis on cells that are selectively expressing
2
DR4 and DR5, mainly killing virus-infected cells and leaving intact normal cells
3
[38,39]. Additionally an antiviral role was proposed for TRAIL. DENV-infected
4
monocytes and dendritic cells display reduced viral replication when TRAIL is
5
exogenously administered [40]. Soluble TRAIL (sTRAIL) was found in sera from
6
dengue patients [41], but mTRAIL role and expression by DENV-2 exposed pDC to has
7
not been investigated yet.
8
In this report we studied pDC activation by DENV and its consequences on viral
9
infection. The clinical study showed that during acute phase of DF, pDCs are activated
10
characterized by TRAIL and IFN-α markers. Indeed, the more pDC are activated the
11
less the disease is severe. We found that DENV-2 efficiently activated TRAIL
12
expression and IFN-α production by pDC. The microscopy study revealed that TRAIL
13
was intracellularly stocked in resting pDC and was relocalized to plasma membrane
14
when pDC were exposed to DENV-2. Furthermore, we showed that pDC could
15
decrease DENV infection in monocytes mainly due to the effects of IFN-α produced.
16
Thus pDC activation constitutes a host defense against DENV-2 infection strongly
17
suggesting that these cells are likely beneficiating the disease outcome.
18
Materials and Methods
19 20
Ethics Statement
21
Experimental procedures with human blood have been approved by Necker Hospital
22
Ethical Committees for human research and were done according to the European
23
Union guidelines and the Declaration of Helsinki. Procedures were also approved by the
24
ethical committee at Instituto de Pesquisas Clinicas Evandro Chagas, FIOCRUZ
25
(CAAE 3723.0.000.009-08). All patients were informed of procedures and gave written
26
consent.
27 28
Patient and blood samples
29
Blood from HIV-1-seronegative blood bank donors was obtained anonymously
30
from “Etablissement Français du Sang” (convention # 07/CABANEL/106), Paris,
31
France. Forty three patients with confirmed dengue fever (Table 1) from two Brazilian
5
1
Health Centers at Campo Grande, MS and Campos de Goytacases, RJ, Brazil were
2
studied. All patients presented clinical diagnosis of dengue infection.
3 4
Criteria for dengue fever severity and laboratorial diagnosis
5
Dengue fever was considered mild when no warning signs (WS) or severe clinical
6
manifestations were observed as follows. Dengue fever with WS was considered if
7
patients presented any of the following warnings: (1) abdominal pain or tenderness; (2)
8
persistent vomiting; (3) Clinical fluid accumulation; (4) mucosal bleeding; (5) lethargy;
9
(6) liver enlargement more than 2 cm associated to laboratory parameters as increase in
10
hematocrit (HCT) concurrent with rapid decrease in platelet counts (hemoconcentration
11
or significant increase in hematocrit together with platelet counts bellow 50,000/mm3).
12
Severe DF was considered if patient displayed fever of 2–7 days plus any of the
13
following: (1) Evidence of plasma leakage, such as high or progressively rising
14
hematocrit evidenced by hemoconcentration; pleural effusions or ascites; circulatory
15
compromise or shock (tachycardia, cold and clammy extremities, capillary refill time
16
greater than three seconds, weak or undetectable pulse, narrow pulse pressure or, in late
17
shock, unrecordable blood pressure); (2) Significant (internal) bleeding. [42,43].
18
Dengue virus infection was confirmed either by anti-dengue-IgM ELISA, serotype
19
specific reverse transcription-polymerase chain reaction (RT-PCR) or by virus isolation
20
as described earlier [44]. Predominant serotypes was Dengue-2 identified in DF±WS
21
(N=10) and Severe DF (N=3) but Dengue-1 was also identified in DF±WS patients
22
(N=6).
23 24
Virus strain and viral stock
25
Dengue virus type 2 (strain Thailand/16681/1984) [45] was used for virus stock
26
preparation as described elsewhere [46]. Briefly, Aedes albopictus cell clone C6/36
27
(CRL-1660, ATCC) were maintained at 28°C in Dulbecco’s modified Eagle Medium
28
(Gibco/Life Technologies, Foster City, CA, USA) with sodium bicarbonate (Sigma-
29
Aldrich, St. Louis, MO, USA) and supplemented with 5% fetal bovine serum (Hyclone,
30
Logan, UT, USA), 1% penicillin-streptomycin-glutamine (Gibco), 0,5% non-essential
31
amino acids (Gibco) and 10% tryptose phosphate broth (Sigma). C6/36 cell monolayers
32
were infected with DENV-2 and cell culture supernatants were harvested 8 days later 6
1
when cytopathic effect was observed. A purified DENV-2 stock was obtained by
2
ultracentrifugation at 100,000g for 1h and set to a final volume 20 times smaller than
3
initial (see also Fig S1) [47,48]. Titration was performed in C6/36 cells using a standard
4
TCID50 (50% tissue culture infective dose) assay as described elsewhere [49].
5
Uninfected flasks were maintained, also purified and used as negative control (MOCK).
6
Infectivity of ultracentrifuged virus inoculum (UC) was comparable with the original
7
C6/36 supernatant (SNDT) because infection rates obtained with the dilution 1/100
8
(UC) is similar to the dilution 1/5 (SNDT) as shown in Fig S1.
9 10
Human cell isolation
11
Cryopreserved peripheral blood mononuclear cells (PBMC) from patients or
12
healthy donors were obtained from density gradient centrifugation of heparinized blood
13
with lymphocyte separation medium (StemCell Technologies, Grenoble, FR). In vitro
14
experiments were performed using fresh PBMC, which were obtained from blood bank
15
donors and isolated as mentioned above. PDCs and monocytes were purified using
16
Human plasmacytoid DC Negative Isolation Kit and Human CD14+ monocytes
17
Isolation Kit, respectively (StemCell Technologies). Cells were cultured in RPMI 1640
18
(Invitrogen, Gaithersburg, MD, USA) containing 10% fetal bovine serum (Hyclone) and
19
1% penicillin-streptomycin-glutamine (Gibco) at 37°C in a humidified 5% CO2 chamber
20
according to protocol.
21
PDC stimulation and coculture with monocytes
22
Freshly purified pDCs were cultured with DENV-2 at approximately MOI 4 to 20,
23
mock for 18 hours (overnight). Chloroquine (Sigma-Aldrich) was used at 5µM/well and
24
added before viral stimulation. Cells were harvested and assessed for pDC cell markers
25
and membrane TRAIL expression or plated on coated slides for 3D microscopy.
26
Supernatant was stored at -70°C for cytokine detection. Monocyte infection was
27
performed as already described [46]. Briefly, freshly isolated monocytes were plated
28
overnight followed by infection with DENV-2 at MOI 10, mock or not infected for 48
29
hours. Soluble human recombinant IFN-α (PBL International, Piscataway, NJ, USA)
30
was added 18 hours before viral infection at 100 IU/mL. For autologous coculture
31
assay, monocytes were cultured overnight in media, meanwhile pDCs were 7
1
chloroquine-treated or not and then stimulated overnight with CpG A 2216 (InvivoGen,
2
San Diego, CA, USA) at 5µM or DENV-2 at MOI 20, or not stimulated. Monocytes
3
were then infected with DENV-2 (MOI 10) and pDCs were added at ratio 1:5
4
pDC/monocytes as explained in Fig. S2. Cells were harvested and assessed for
5
intracellular DENV antigens.
6
Flow cytometry
7
Antibodies for fluorescein isothiocyanate (FITC)-conjugated anti-CD123 or
8
BDCA-2 (Miltenyi Biotec, Auburn, CA), Phycoerythrin (PE)- conjugated CD11c
9
(IOTest/Beckman Coulter, Marseille, FR), Allophycocyanin (APC)-conjugated anti-
10
BDCA-4 (Miltenyi Biotec) and Allophycocyanin-Cy7 (APC-Cy7)-conjugated anti-
11
CD14 (BD Biosciences, San Jose, CA), Vioblue-conjugated anti-CD4 (Miltenyi
12
Biotec), V500 anti-CD3 (BD Biosciences) or with appropriate isotype-matched control
13
antibodies (at 5 mg/mL each) in PBS containing 2% fetal bovine serum (Hyclone) and
14
2mM EDTA (Gibco). Human PBMCs or isolated monocytes/pDCs were incubated for
15
20 min at 4°C with antibody cocktails. Cells were washed twice in ice-cold PBS and
16
flow cytometry acquisition was performed on FACSCanto 7 colors or FACS Aria 13
17
colors flow cytometers using FACSDiva software (BD Biosciences). CD3- CD4+ CD14-
18
CD123+/BDCA-2+ BDCA-4+ gated cells were then tested for the expression of surface
19
markers using PE-labeled anti-TRAIL (BD Biosciences). Mosquito C6/36 cell line
20
monolayers were washed with PBS-1% bovine serum albumin (Sigma) and incubated
21
for 60 min at 4°C with purified anti-DENV-complex (Millipore, Billerica, MA, USA)
22
then 30 min with goat anti-mouse Alexafluor647 (Molecular Probes/Life Technologies)
23
and fixed. Intracellular antigen staining for C6/36 or cocultures was performed using
24
2% paraformaldehyde (Sigma) followed by antibodies staining steps with 0,1% saponin
25
(Sigma) buffer. Cells were analyzed by C6 Cytometer (Accuri/BD Biosciences). FlowJo
26
software (Treestar, Ashland, OR, USA) was used to analyze flow cytometry data.
27
Three dimension (3D) microscopy and immunofluorescence
28
Cells were plated on poly-L-lysine (Sigma)-coated slides and then fixed in 4%
29
paraformaldehyde (Sigma), quenched with 0.1 M glycine (Sigma). Cells were blocked
30
and incubated in permeabilizing buffer containing 0.1% saponin (Sigma) with mouse
31
anti-TRAIL (clone RIK-2, eBioscience, San Diego, CA) or mouse anti-DENV (clone 8
1
D3-2H2-9-21, Millipore). TRAIL and DENV staining were revealed using a secondary
2
donkey anti-mouse IgG-Cy3 (Jackson ImmunoResearch, West Grove, PA, USA).
3
Nucleus was stained using DAPI (Molecular Probes/Life Technologies). Mounted slides
4
were scanned with a Nikon Eclipse 90i Upright microscope (Nikon Instruments Europe,
5
Badhoevedorp, The Netherlands) using a 100x Plan Apo VC piezo objective (NA 1.4)
6
and Chroma bloc filters (ET-DAPI, ET-Cy3) and were subsequently deconvoluted with
7
a Meinel algorithm and 8 iterations and analyzed using Metamorph (MDS Analytical
8
Technologies, Winnersh, UK). Overlays were: TRAIL or DENV / DAPI / Trans.
9
ImageJ (NIH, Bethesda, MD, USA) plugin 3D interactive surface plot was used on
10
overlay stack on pDC stained with TRAIL or DENV/DAPI. Quantity of TRAIL and
11
DENV-2 were determined using the measure and label plugin (ImageJ). C6/36
12
mosquito cell line were plated on slides and fixed with cold acetone. Mosquito cells
13
were stained with mouse anti-DENV complex (Millipore) in PBS-1%bovine serum
14
albumin (Sigma), washed twice with PBS. DENV E protein was revealed with goat
15
anti-mouse Alexafluor488 (Molecular Probes/Life Technologies). Slides were mounted
16
with ProLong Gold with DAPI (Molecular Probes/Life Technologies) and visualized at
17
Evosfl Microscope (AMG, Bothell, WA, USA).
18 19
Cytokine detection
20
Supernatants of pDCs/monocytes or cocultures in presence of DENV-2 or
21
negative controls as well as acute phase plasma from dengue patients were tested for
22
multispecies soluble IFN- by ELISA (PBL International) according to the
23
manufacturer’s instructions. Plasma samples were also tested for soluble TRAIL by
24
ELISA (R&D Systems, Minneapolis, MN, USA)
25 26
Statistical analysis
27
Experiments were repeated at least four times. P values (P) were determined using
28
a two-tailed Student’s t test for in vitro data and nonparametric Mann-Whitney test for
29
patient data. P
