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. Author manuscript; available in PMC: 2017 Sep 1.
Published in final edited form as: Virology. 2016 Jun 24;496:227–236. doi: 10.1016/j.virol.2016.06.008

Dengue Virus NS1 Enhances Viral Replication and Pro-Inflammatory Cytokine Production in Human Dendritic Cells

Farah Alayli 1, Frank Scholle 1,#
PMCID: PMC4969143  NIHMSID: NIHMS800662  PMID: 27348054

Abstract

Dengue virus (DV) has become the most prevalent arthropod borne virus due to globalization and climate change. It targets dendritic cells during infection and leads to production of pro-inflammatory cytokines and chemokines. Several DV non-structural proteins (NS) modulate activation of human dendritic cells. We investigated the effect of DV NS1 on human monocyte- derived dendritic cells (mo-DCs) during dengue infection. NS1 is secreted into the serum of infected individuals where it interacts with various immune mediators and cell types. We purified secreted DV1 NS1 from supernatants of 293T cells that over-express the protein. Upon incubation with mo-DCs, we observed NS1 uptake and enhancement of early DV1 replication. As a consequence, mo-DCs that were pre-exposed to NS1 produced more pro-inflammatory cytokines in response to subsequent DV infection compared to DCs exposed to heat-inactivated NS1 (HNS1). Therefore the presence of exogenous NS1 is able to modulate dengue infection in mo-DCs.

Keywords: Dengue, NS1, nonstructural protein NS1, dendritic cells, proinflammatory cytokines, immune modulation

INTRODUCTION

Dengue virus (DV) is a member of the family Flaviviridae and genus Flavivirus. It is the most prevalent arboviral infection worldwide with approximately 50-100 million reported infections annually (WHO). DV is transmitted to humans through the mosquito vectors Aedes albopictus and Aedes aegypti. These vectors are mainly found in the tropical and subtropical regions of the world with their geographic region expanding (Van Kleef et al., 2010), potentially exposing 40% of the world's population to dengue infection (WHO). There are four antigenically distinct but closely related serotypes of DV (DV1-4) (Halstead, 1988). Upon primary infection with one of the serotypes, symptoms typically range from subclinical to self-limiting dengue fever. Upon secondary infection with a heterologous serotype, serotype cross-reactive antibodies developed during primary infection increase the risk of developing dengue hemorrhagic fever (DHF) or dengue shock syndrome (DSS) in a process termed antibody-dependent enhancement (ADE) (Halstead and O'Rourke, 1977). During ADE, sub-neutralizing antibodies enhance the infection of Fc receptor bearing cells leading to increased viremia and a subsequent cytokine storm which are thought to contribute to the manifestation of severe disease (Goncalvez et al., 2007, Guzman et al., 2013, Kliks et al., 1988, Rothman, 2011).

Dengue virus has a single stranded, positive sense, 11kb RNA genome with a single open reading frame. It is an enveloped virus, and upon release into the cytoplasm, the genome serves as mRNA and is directly translated into a single polyprotein. It is cleaved co- and post-translationally by host and viral proteases into three structural (C, prM, and E), and seven non-structural (NS) proteins (NS1, NS2a, NS2b, NS3, NS4a, NS4b, NS5). Dengue non-structural protein 1 (NS1) is a 46 kD glycoprotein that exists within the infected cell, cell surface associated, and secreted into the blood stream (Flamand et al., 1999, Winkler et al., 1989, Young et al., 2000a). Upon translation, flavivirus NS1 translocates into the lumen of the ER where it dimerizes and is thought to play a structural role in the replication complex of the virus by interacting with NS4B (Youn et al., 2012). Studies also show that NS1 plays a vital role in early negative strand viral replication (Lindenbach and Rice, 1997, Lindenbach and Rice, 1999, Mackenzie et al., 1996). However, the exact mechanism of NS1's role in viral replication remains elusive.

DV NS1 is secreted as an oligomer, which serves as a major immunogen during the acute phase of infection leading to a strong anti-NS1 humoral response. Secreted DV NS1 has been implicated with both protective and immunopathogenic roles. It was initially identified as a complement fixing protein in the blood (Chambers et al., 1990). Recent studies show that NS1 leads to the activation of complement and contributes to endothelial cell damage (Kurosu et al., 2007), whereas other studies report that NS1 prevents complement activation which serves as an immune evasion strategy protecting DV particles from complement-mediated lysis (Avirutnan et al., 2011). Anti-NS1 antibodies have been shown to provide protective immunity against lethal dengue challenge in mice (Beatty et al., 2013, Huang et al., 2013, Wu et al., 2003), and other studies report auto-reactivity with blood clotting proteins (Lin et al., 2012, Sun et al., 2007) and endothelial cells (Liu et al., 2011, Modhiran et al., 2015). Furthermore, anti-NS1 antibodies bind to NS1 on the surface of endothelial cells leading to iNOS mediated apoptosis (Lin et al., 2002), or potentially targeting the endothelial cells for complement mediated lysis and contributing to endothelial cell damage and severe disease (Avirutnan et al., 2006).

Dendritic cells (DCs) are sentinels and bridge the innate and adaptive immune responses during viral infections (Steinman and Banchereau, 2007). Immature DCs are a primary target for DV upon injection into the skin (Marovich et al., 2001, Schmid et al., 2014, Tassaneetrithep et al., 2003). Once infected with DV, DCs upregulate various co-stimulatory molecules and pro-inflammatory cytokines to initiate the anti-viral response (Ho et al., 2001, Libraty et al., 2001). However, it has been shown that various NS proteins modulate type I IFN signaling and production in human DCs (Mazzon et al., 2009, Morrison et al., 2013, Munoz-Jordan et al., 2005, Rodriguez-Madoz et al., 2010a, Rodriguez-Madoz et al., 2010b). Studying the effect that DV NS1 may have on DCs will contribute to our understanding of dengue immunopathogenesis for the development of anti-dengue therapeutics and vaccines.

Previously, investigators in our lab group have has shown that intracellularly expressed and secreted West Nile virus NS1 inhibits TLR3 mediated production of IL-6 in HeLa cells, as well as mouse bone marrow-derived dendritic cells and macrophages (Crook et al., 2014, Wilson et al., 2008). Similar studies were carried out with DV1 NS1 and human monocyte derived DCs (mo-DCs), however, we were not able to detect an effect on TLR3 signaling (data not shown). Therefore, we investigated whether DV NS1 modulates DC activation during dengue infection. To our knowledge, this is the first study to directly investigate whether soluble NS1 interacts with human DCs and whether it modulates their response during DV infection.

MATERIALS AND METHODS

Cell Lines

Full-length dengue serotype 1 NS1 plus the last 30 amino acids of E protein, encoding the signal sequence, and a C-terminal HA tag were cloned into the pLEX-MCS lentiviral vector. It was cotransfected into HEK-293T cells along with packaging (pREV and pMDLg), and envelope (pVSV-G) plasmids as described previously (Crook et al., 2014). Lentivirus containing supernatants collected at 40h post trasfection, and used to transduce HEK-293T cells. Transduction was followed by selection with 4μg/ml of puromycin, to create cell lines that stably express and secrete DV1 NS1-HA protein. Empty vector control (pLEX-MCS) lentiviruses were used to create 293T-MCS cells that served as a negative control for protein expression and purification. Immunostaining using anti-HA primary antibody (Sigma H3663) and HRP conjugated goat anti-mouse secondary antibody were used to detect the expression of NS1. Secretion of NS1 from the 293T cell lines was confirmed by Western blot using anti-NS1 monoclonal antibody (data not shown).

NS1 purification

293T-NS1 and 293T-MCS cells were grown to confluency; cell free supernatants were collected and used for the immuno-purification of NS1 using anti-NS1 mAb clone 5.7.9 (provided by Mary-Ann Accavitti-Loper, SERCEB antibody production Core, University of Alabama at Birmingham). 293T-MCS cell line supernatants were treated identically to the 239T-NS1 supernatants during the purification process and served as the purification control. Mouse monoclonal anti-NS1 antibody was coupled to cyanogen bromide (CNBr)- Activated Sepharose 4B beads (GE 17-0430-01) and the purification was carried out based on the manufacturer's instructions. Briefly, cell free supernatants were collected from 293TNS1/239T-MCS cell lines and passed through a column packed with antibody-coated beads. The column was washed with a neutral pH buffer, and a high pH (11.2) elution buffer was used to elute NS1 from the beads. 1 ml fractions were collected neutralized with a Tris-HCl buffer (pH 7), and NS1 ELISA was performed to quantify NS1 in the fractions. Fractions containing NS1 were pooled and concentrated using dialysis tubing and PEG 20,000, then dialyzed against PBS overnight at 4°C. Protein concentration was determined using a nanodrop spectrophometer and confirmed using Bradford Assay (BioRad). Protein purity was determined by silver staining of SDS-PAGE gels and compared to the MCS purified fraction. NS1 was heat denatured (HNS1) at 95°C for 1.5h which was used as a negative control.

Virus Propagation

WHO reference strain dengue virus type 1 (DV1) (West Pacific 74) was obtained from Dr. Aravinda de Silva, UNC Chapel Hill. DV1 was grown and propagated in the Aedes albopictus C6/36 mosquito cell line at 28°C with 5% CO2. C6/36 cells seeded in T-150 flask and at 75% confluency were infected at MOI 0.01 DV1 with 5 ml of C6/36 media consisting of MEM, 10% FBS-HI, and 10% tryptose phosphate broth. Viral attachment was allowed to occur for 1h with rocking every 15 minutes, and then an additional 10 ml of media were added and allowed to incubate for 7 days. The supernatant was then clarified by centrifugation in preparation for purification.

Virus Purification

Virus purification was performed using a discontinuous OptiPrep gradient (25%, 30%, 35%, 45%, 50%). 10 ml of cell free viral supernatant were overlaid onto the gradient, and centrifuged at 24,000 RPM for 2.5 hours at 4°C. Half milliliter fractions were collected from the bottom and assessed for the presence or absence of NS1 using Western Blot and anti-NS1 antibody. Virus-containing fractions that were NS1 free were pooled and passed through an Amicon Ultra Centrifugal Filter (UFC910008) and washed 4 times with PBS, and then resuspended in MEM supplemented with 20% FBS, 1% Penicillin/Streptomycin, and 20 mM HEPES buffer. The virus was stored at 0.5 ml aliquots at −80°C until use. Purified virus was used for all experiments.

Viral Quantification

Viral titer was determined by immunofocus formation assay on Vero cells and expressed in ffu/ml. Briefly, Vero cells were plated to confluency in 48 well plates, virus was allowed to attach for 1h with rocking every 15 minutes at 37°C, then a 1:1 ratio of tragacanth gum/MEM (with 1% HEPES, 1% Pen/Strep, 1% FBS) mixture was overlaid on the cells and allowed to incubate for 48h. Semisolid mdium was removed and the cells were washed with PBS and fixed with 1:1 ratio of acetone to methanol for 15 minutes at −20°C. The cells were then blocked with 1% normal horse serum (NHS)/PBS solution and probed with D1-4G2 (anti-E) antibody for 1h followed by goat anti-mouse HRP secondary antibody for 1h at room temperature. Immunofoci were detected using Vector VIP kit (Vector Labs SK-4600), and foci were counted using an inverted microscope to calculate ffu/ml (focus forming units/ml).

Generation of monocyte derived DCs (mo-DCs)

Whole blood (50-60 ml) from healthy human donors was obtained from Gulf Coast Regional Blood Center in Houston, TX. Using a Ficoll-Paque PLUS gradient (GE Healthcare), the PBMC layer was isolated, the cells were washed and cryopreserved at 5×106 cells/ml in 90% FBS-HI, 10% DMSO. To differentiate DCs, cryopreserved PBMCs were thawed, and monocytes were allowed to adhere to tissue culture plastic flasks (TPP) for 90 minutes at 37°C, non-adherent cells were removed by gentle rocking and aspiration. Adherent monocytes were washed with PBS twice and then replenished with complete PBMC media for 7 days with the addition of 800 IU/ml rhGMCSF and 400 IU/ml rhIL-4 (R&D systems). PBMC media consisted of RPMI, 10% FBS-HI, 1% Penicillin/Streptomycin, 1% non-essential amino acids. The cells were incubated in a 37°C incubator with 5% CO2, and 95% relative humidity. After 5-7 days, immature non-adherent mo-DCs were collected and their phenotype was assessed using CD14-FITC, CD86-PE, HLA-DR-APC, CD11c-APC, CD1a-APC, DCSIGN-FITC, CD80-FITC, CD86-PE antibodies or the corresponding isotype controls (eBioscience). An Accuri C6 Flow Cytometer (BD) was used to assess the surface expression of the mo-DC markers.

Infections

Mo-DCs were seeded into a 96 well plate (Corning 353872) at 1×105cells/well, and 20 μg/ml purified NS1 or HNS1 was added for 16h. Mo-DCs were then washed once with PBS and resuspended in complete PBMC media, purified virus was added for 1.5 hours at MOI 1. At this MOI approximately 85% of moDCs subsequently stained positive of DV1. After viral attachment for 1.5h, the cells were washed twice with PBS and resuspended in complete PBMC media for the remainder of the infection time; 24h unless otherwise noted. mo-DC concentration was maintained at 1×106 cells/ml for all experiments.

Flow Cytometry

To determine percentage of infected cells, mo-DCs were harvested and washed with PBS, then fixed and permeabilized using BD Cytofix/Cytoperm kit (BD 554714). The cells were then incubated with anti-E antibody D14-G2 at 1:1000 dilution, followed by goat anti-mouse Alexa-fluor AF488 -conjugated antibody. The number of infected cells was determined using C6 Accuri Flow Cytometer (BD).

Real-time RT-PCR

Mo-DCs were infected as described above, at the corresponding time points the cells were washed with PBS twice, then lysed using RLT buffer from the RNeasy Qiagen Kit. Lysates were then homogenized using QIAshredder columns (Qiagen). Total RNA was purified according to the manufacturer's instructions and quantified using a nanodrop spectrophotometer. cDNA was synthesized using Impromp II Reverse Transcription kit (Promega), and random hexamers as primers. Real-time PCR was then carried out to quantify GAPDH and DV1-RNA copies using sensiFAST SYBR green (Bioline) and the following primers:

GAPDH: Forward: 5’GGATTTGGTCGTATTGGGCG-3’
Reverse: 5’-TGGAAGATGGTGATGGGATTTC-3’
NS5: Forward: 5’-GCGGTTCTGGGACCTTGTGC-3’,
Reverse: 5’-AGAAAGCGTGCTCCCAACCACA-3’
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NS5 copies per 106 GAPDH copies were determined using standard curves for both genes as described in (Wilson et al., 2008). For the qPCR array, we used the SA biosciences RT2 Profiler PCR array for Human Dendritic & Antigen Presenting Cells (PAHS-406ZA) and processed the RNA per the manufacturer's instructions. The ΔΔCt method was used to calculate fold induction of the infected NS1 samples compared to uninfected NS1 and infected HNS1 to uninfected HNS1. Positive values indicate gene upregulation and negative values indicate gene down-regulation.

ELISA

Mo-DCs pretreated with NS1/HNS1 were infected with DV1 for 24h and supernatant was collected. CCL2 and IL-6 levels were quantified using ELISA kits (eBioscience) according to the manufacturer's instructions.

Statistical Analysis

Statistical analysis was performed using nonparametric student t-test. Statistical significance determination: p≤0.05 *, p≤0.0005**, p≤0.0001***. Error bars represent standard error of the mean (SEM).

RESULTS

Purified dengue virus serotype 1 NS1 is taken up by immature mo-DCs in vitro

NS1 is secreted into the serum of patients infected with DV and studies show that the level of NS1 in the blood stream may correlate with severity of disease (Libraty et al., 2002a, Vaughn et al., 2000). Since dendritic cells are the primary target for dengue virus infection in vivo, we were interested in assessing a potential effect of dengue virus type 1 (DV1) soluble NS1 on dendritic cells during infection. To this end, the interaction of soluble NS1 with human monocyte-derived dendritic cells (mo-DCs) was first investigated. Full length NS1 was over-expressed in 293T cells using a lentivirus system. MCS empty vector lentivirus transduced into 293T cells served as a negative control. Expression of NS1 in the 293T-NS1 cells was first confirmed by immunostain, and secretion of NS1 into the supernatant was confirmed by Western blot analysis (data not shown). Secreted NS1 from 293T serum-free supernatant was then purified using immunoaffinity purification using DV1 NS1 antibody. NS1 fractions were eluted, and their NS1 content was analyzed by NS1-specific ELISA (Figure 1A). Fractions containing NS1 were pooled and purity was assessed by SDS-PAGE and silver stain. As a control, supernatants from MCS-293T cells (MCS-Ctrl) were treated and processed identically as 293T-NS1 supernatants. The monomer, dimer, and hexamer forms of NS1 were readily detected in the silver stain gel (Figure 1B). Next, we assessed NS1 uptake by immature human mo-DCs in vitro. To investigate both an early and a later time point of potential interaction of NS1 with mo-DCs, mo-DCs were incubated with varying concentrations of purified NS1 or equal volumes of MCS-Ctrl at two different time points (6h and 16h) at 37°C. After treatment, the mo-DCs were fixed and permeabilized, and NS1 uptake was assessed by flow cytometry using an anti-NS1 antibody. At 6h, mo-DCs had internalized NS1 in a dose dependent manner although only very little uptake could be detected (Supplemental Figure 1). By 16h at an NS1 concentration of 20μg/ml and above the majority of cells were NS1 positive compared to cells treated with the MCS-Ctrl (Figure 2A). At later times, the amount of NS1 within the cells decreased (data not shown), presumably by degradation within endosomes. To determine whether NS1 was bound to the cell surface or became endocytosed DCs that either fixed and permeabilized or fixed without permeabilization were compared when incubated with 20 μg/ml of NS1. NS1 positive cells were only detected in the permeabilized cells (Fig 2B). These results demonstrate that soluble NS1 is able to interact with and become endocytosed by human mo-DCs.

Figure 1. NS1 purification.

Figure 1

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A. Immuno-purified NS1 fractions analyzed by ELISA using anti-NS1 monoclonal antibody. MCS denotes supernatants from non-NS1 expressing cells undergoing an identical purification procedure. B. 1 μg purified NS1 or same volume MCS purification control were analyzed for purity on a silver stained SDS-PAGE gel.

Figure 2. NS1 uptake by mo-DCs.

Figure 2

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A. 2×105 immature mo-DCs were incubated with 5, 10, 20, 30, 40 μg/ml purified NS1 or the same volume of MCS-ctrl. After 16h, cells were harvested, fixed and permeabilized, then anti-NS1 primary Ab and anti-mouse AF-488-conjugated secondary antibodies were used to assess NS1 uptake by flow cytometry. Cells were analyzed on BD Accuri C6 Flow cytometer. B) After incubation with 20 μg/ml of NS1 for 16h, mo-DCs were either permeabilized or kept nonpermeabilized prior to staining with anti-NS1 antibody, Data are representative of three independent experiments, and similar NS1 uptake was verified for all donors used.

Pre-exposure to DV1 NS1 does not lead to mo-DC maturation

After confirming NS1 uptake by mo-DCs, we analyzed the consequences of this on expression of normal and co-stimulatory DC markers. Surface expression of HLA-DR, DC-SIGN, CD86, and CD80 was used to assess the phenotype of the mo-DCs. To make sure mo-DCs were responding appropriately, stimulation with pIC and LPS in comparison to DV infection was carried out and secretion of IL-6 and upregulation of CD86 was monitored (Supplemental Figure 3). Activation of mo-DCs by DV was weaker than with pIC or LPS stimulation but easily detectable nonetheless. Immature mo-DCs were incubated with 20 μg/ml of purified NS1 or an equal amount of heat-inactivated NS1 (HNS1) for 16h at 37°C.. After 16h, NS1 treatment alone did not alter the expression of these markers compared to cells treated with HNS1 (Figure 3, top panel). Next, we investigated the effect of NS1 pretreatment of mo-DCs on their expression of the same markers during DV infection. In most published studies, researchers use clarified supernatants from DV infected C6/36 cells for infections. However, NS1 is found in the C6/36 viral supernatant that is typically used for infections (data not shown), which may alter results that might be obtained with purified virus. Therefore, we purified DV1 from the clarified C6/36 supernatants using ultracentrifugation, and only used fractions that were NS1 free (data not shown). After a 16h treatment of mo-DCs with NS1/HNS1 followed by 24h of DV1 infection at an MOI of 1, we found that surface expression of DC-SIGN, HLA-DR, and CD80 was unchanged compared to uninfected mo-DCs, whereas, levels of CD86 were equally upregulated in both the NS1 and HNS1 treated groups (Figure 3, bottom panel). As an additional control we also compared cell surface marker expression on mo-DCs when treated with either MCS control, HNS1 and NS1. In either case the expression of each marker was very similar (Supplemental Figure 2). These data suggest that NS1 treatment alone does not alter the expression of DC-SIGN, HLA-DR, CD80 and CD86 in uninfected mo-DCs, and does not influence it during DV infection.

Figure 3. mo-DC phenotype is unaltered after NS1 exposure compared to HNS1.

Figure 3

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A. mo-DCs were treated with 20 μg/ml NS1 or HNS1 for 16h, the cells were washed once with PBS. Surface expression of HLA-DR, DC-SIGN, CD80 and CD86 were determined using specific antibodies or their isotype controls (eBioscience) and assessed by flow cytometry. NS1 (blue) pretreated cells and HNS1 (orange) pretreated cells were plotted for all markers tested and compared to isotype control (tinted red). B. mo-DCs were pretreated with NS1/HNS1 at 20 μg/ml for 16h, then washed and resuspended with fresh PBMC media and purified DV at MOI 1. Viral attachment was allowed for 1.5h, cells were washed to remove excess virus, then resuspended with fresh PBMC media for 24h. After 24h the cells were analyzed for the markers as in part A. Data are representative of three independent experiments using three different donors.

NS1 leads to enhanced DV1 infection of mo-DCs

It has been reported that DV1 NS1 is endocytosed by hepatocytes leading to enhanced endocytic activity and subsequent virus production (Alcon-LePoder et al., 2005). We hypothesized that DV1 NS1 may have a similar effect on mo-DCs. To test this hypothesis, mo-DCs were pretreated with NS1 or HNS1 as previously described, followed by DV1 infection at an MOI of 0.1 for 24h. The cells were then assessed for their infection status via flow cytometry using the D1-4G2 anti-E antibody. As a result of NS1 pre-exposure, a significantly higher percentage of mo-DCs were infected compared to mo-DCs pre-exposed to HNS1 (Figure 4A). We further confirmed this effect by quantifying DV1 genome copy number within the cells by qRT-PCR. mo-DCs were infected at MOI 0.5 for 6h, 8h, and 24h and total RNA was purified and used to quantify genome copies by amplification of part of the NS5 coding sequence by qRT-PCR. A statistically significant enhancement of DV1 genome copies was observed at 8h in NS1 pretreated compared to HNS1 pretreated mo-DCs (Figure 4B). By 24h, the difference subsided. This indicates that increased DC infectability and subsequent viral RNA replication after NS1 pre-exposure are early phenomena that might serve to give the virus an early replication advantage. Together, these data provide evidence that NS1 uptake by DCs influences their susceptibility to DV infection, and leads to enhanced viral replication.

Figure 4. NS1 leads to a higher percentage of infected DCs and enhances early dengue replication.

Figure 4

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mo-DCs were pretreated with NS1/HNS1 (20 μg/ml for 16h). A. The mo-DCs were then infected at MOI 0.1 for 24h, anti-E primary antibody D1-4G2 and anti-mouse AF-488 secondary Ab were used to assess the percentage of infected cells by flow cytometry. The number of infected DCs is expressed in percent compared to an uninfected control. The data shown are an average of 3 experiments with 3 different donors. B. After NS1 pretreatment (as before), mo-DCs were infected at MOI 0.5, then total RNA was harvested after 6h, 8h, and 24h. cDNA synthesis was carried out, and qRT- PCR specific for a region within the NS5 coding sequence was used to quantify genome copy number per 106 GAPDH copies. Data are representative of three independent experiments using three different donors.

Enhanced mo-DC infectability by NS1 leads to enhanced pro-inflammatory cytokine production during DV1 infection

Although pretreatment of mo-DCs with DV1 NS1 did not lead to alteration of maturation molecules during infection, it was possible that enhanced replication would lead to enhanced pro-inflammatory cytokine production. To test this hypothesis, mo-DCs were pretreated with NS1 or HNS1 (20ug/ml for 16h), and then infected with DV1 at an MOI of 1 for 24h. Total RNA was extracted and quantified using a Real-time PCR array specific for Dendritic & Antigen Presenting Cells (SA Biosciences). Upon pre-exposure to NS1/HNS1 without infection, there were only 7 out of 84 genes that were up or down regulated two fold or more between the two treatment groups. The upregulated genes included CCR2, CD1C, and IL-12B (IL12-p40) and the downregulated genes were CCL7, CXCL12, FcεR2, and TLR7 compared to the HNS1 pretreated uninfected mo-DCs (Figure 5A and Table 1). Upon additional DV1 infection for 24h, there were 10 genes that were upregulated 25 fold or more over mock infected cells, regardless of NS1 or HNS1 pretreatment. However, in this group, NS1 pretreatment followed by infection led to heightened upregulation of CCL2, IL-6, CXCL10, IL12A (IL-12-p35), and TNFα compared to mo-DCs that were pretreated with HNS1 and infected (Figure 5B). Among the genes that were upregulated to a lower level, we detected 18 genes that were upregulated between 5 and 20 fold over mock in the two treatment groups upon infection, and NS1 pretreatment enhanced induction of 8 of these genes compared to the HNS1 pretreated mo-DCs (Figure 5C). Additionally, there were 20 down regulated genes as a result of infection, and cells pretreated with NS1 expressed lower levels of these 15 genes including IL-16, FCER1A, FCER2, IL-10, and ITGAM compared to the HNS1 group (Figure 5D). These data indicate that DCs pre-exposed to NS1 are generally more activated following DV1 infection compared to DCs pre-exposed to the heat-inactivated form (HNS1).

Figure 5. NS1 uptake followed by DV1 infection leads to enhanced pro-inflammatory cytokine production by mo-DCs.

Figure 5

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moDCs were pre-treated witih NS1/HNS1 (20 μg/ml for 16h), washed once with PBS, then infected for 24h with DV1 at MOI 1 as described previously. After 24h, RNA was collected and cDNA was synthesized per manufacturer's instructions and quantified using RT2 PCR array. Gene values were normalized to house-keeping genes and fold induction (over mock) was calculated by ΔΔCt method comparing NS1 infected to NS1 mock and HNS1 infected to HNS1 mock. (A) Fold induction of NS1 mock vs. HNS1 mock. Genes were grouped by level of upregulation >50 fold (B) 5-20 fold (C), and down-regulation (D). Data are representative of two independent experiments using two different donors.

Table 1.

Fold induction Dendritic and Antigen Presenting Cell Array Infected vs. Uninfected

Gene NS1 infect vs. NS1 mock HNS1 infect vs. HNS1 mock
CCL19 21.2590 8.2249
CCL2 190.0190 101.8287
CCL3 15.8895 11.9588
CCL5 99.7331 144.0075
CCL7 40.7859 19.2929
CXCL1 40.7859 44.3235
IL12A 225.9720 103.9683
IL6 105.4197 56.4930
TNF-a 91.1392 69.0706
CXCL10 9152.8160 6517.0350
IL12B 9.0005 19.2929
CCL13 4.5631 6.4086
CD40 9.0631 9.7811
CD80 9.0631 9.1896
CSF2 4.4383 3.6301
CCL8 1.3300 2.0279
VCAM1 1.1900 2.8679
IRF8 2.2815 1.6400
LYN 2.2501 1.8300
TLR7 3.5554 −1.2900
FCGR1A 3.2043 −7.7800
CXCL2 4.7568 4.5315
FAS 6.0629 2.1435
ICAM1 2.3457 3.2266
IL8 6.1050 4.3469
IRF7 7.6741 6.4531
NFKB1 2.0994 2.9079
TAP2 3.4105 3.4105
CD4 −3.5801 −5.4264
CD74 −2.0000 −2.5315
CEBPA −5.2780 −2.8089
CSF1R −3.4105 −3.8637
CXCR4 −2.9282 −3.7321
HLA-DMA −3.0314 −3.3404
HLA-DPA1 −2.9690 −2.7702
IL16 −4.7899 −7.5162
ITGB2 −2.0849 −2.6390
LRP1 −2.3950 −2.6945
THBS1 −6.2333 −6.5887
RPLP0 −2.4116 −2.6759
FCER1A −1.7800 −7.7812
FCER2 1.0100 −3.2716
IL10 1.7300 −2.2501
ITGAM −1.6500 −2.3134
PTPRC −1.9900 −2.6027
RAC1 −1.6100 −2.3620
TLR1 −1.6800 −2.2191
TLR2 −1.3700 −2.6208
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Dark shaded: Genes upegulated >25 fold, Light shaded: genes upregulated 5-20 fold, non-shaded: downregulated genes,

NS1 pre-treated mo-DCs produce more IL-6 and CCL-2 during DV infection

Next, we confirmed that some of the observed differences in transcript levels were translated to differences in secreted protein. Supernatants from mo-DCs pretreated with NS1/HNS1 and infected with DV1 for 24h were collected and analyzed by ELISA for CCL2 and IL-6 protein content (Figure 6). We chose these two molecules because their transcript levels were notably different between NS1 and HNS1 pretreated/infected mo-DCs and they were both upregulated 50 fold or more over mock infected cells. Production of both CCL2 and IL-6 protein was significantly enhanced in NS1 pretreated cells consistent with the PCR array data. Based on these results, we conclude that NS1 pretreated DCs are more activated during infection as a result of enhanced early viral replication.

Figure 6. Enhanced IL-6 and CCL2 production by NS1 pretreated DV1 infected mo-DCs.

Figure 6

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mo-DCs were pretreated with NS1/HNS1 (20 μg/ml 16h), then infected with DV1 at MOI 1 for 24h. Supernatants were harvested, and IL-6 and CCL2 secretion levels were analyzed by ELISA and expressed in pg/ml. OD450 was measured using spectrophotometer and protein levels were calculated based on a standard curve provided by the kit. This is an average of two experiments from two different donors.

DISCUSSION

NS1 is secreted to high levels in the sera of infected patients (Alcon et al., 2002), and high concentrations of NS1 in the blood have typically been associated with more severe disease (Avirutnan et al., 2006, Hang et al., 2009, Libraty et al., 2002b, Young et al., 2000b). Secreted DV NS1 (NS1) associates with various cell types including epithelial cells, fibroblasts, hepatocytes, and certain endothelial cell types (Alcon-LePoder et al., 2005, Avirutnan et al., 2007). To date, there are no published studies assessing whether NS1 directly associates with DCs; the primary target cell for DV infection. Using purified DV1 NS1, we demonstrated that NS1 associates with, and is taken up by human mo-DCs as early as 6h post infection (Figure 2). Since DCs are professional antigen presenting cells, it was possible that NS1 uptake may modulate the immature phenotype of mo-DCs, but that was not the case when we assessed expression of HLA-DR, DCSIGN, CD80, and CD86 after 16h treatment with NS1/HNS1. Upon DV1 infection for 24h, HLA-DR, DCSIGN, and CD80 levels did not change compared to uninfected mo-DCs, whereas CD86 was upregulated equally in the NS1 and the HNS1 pretreated mo-DCs (Figure 3). This result indicates that DV infection does not alter expression of HLA-DR, DCSIGN, and CD80 but does allow upregulation of CD86, and that NS1 does not play a role in modulating this effect.

Studies have shown that DV NS1 plays a role in early negative strand viral replication and that it co-localizes with dsRNA (MACKENZIE et al., 1996, Muylaert et al., 1996, Westaway and Blok, 1997). Upon trans-complementation, NS1 can rescue replication of defective flaviviral genomes (Khromykh et al., 1999, Lindenbach and Rice, 1997, Morrison and Scholle, 2014). Although these studies indicate a vital role for DV NS1 during replication, the exact mechanism remains elusive. Alcon-LePoder et al. (Alcon-LePoder et al., 2005) demonstrated that DV1 NS1 may alter the physiology of the hepatocytes by enhancing their endocytic activity leading to more viral production. In this study NS1 increased the endocytic activity of hepatocytes non-specifically as illustrated by increased uptake of rhodamine-labeled dextran. In light of these studies, we hypothesized that NS1 pretreatment of mo-DCs may have a similar consequence. We found that upon pretreatment of mo-DCs with NS1, there was a significantly higher percentage of infected mo-DCs at 24 hours post DV1 infection compared to mo-DCs pretreated with HNS1. When we investigated viral replication by measuring genome copy number, we found that NS1 pretreatment enhanced DV1 RNA replication only early during replication (8h but not 24h) (Figure 4). To assess whether increased RNA replication translated to increased virus output, viral production was measured at 16h and 24h post infection using an immunofocus assay (data not shown). At 16 hours, no virus was detected and at 24h we were not able to detect a difference in DV production between the NS1 pretreated and HNS1 pretreated DV1 infected mo-DCs. Therefore, in this in vitro culture system with isolated moDCs, increased initial viral RNA replication did not result in increased virus production. It is conceivable however, that the interplay of enhanced infectability in the presence of NS1, altered cytokine production and the presence of several different cell types could generate a milieu conducive for an increase in virus production in vivo.

Enhanced dengue infection has been previously described in the context of ADE where sub-neutralizing antibodies mediate enhanced infectivity of FcR bearing cells. This phenomenon usually results in enhanced viremia and an immune mediated cytokine storm which is thought to be the main contributor to severe disease. Our data suggest that NS1 is enhancing infectability of DCs. Upon assessment of the downstream effect of enhanced viral replication, we found a marked increase in DC pro-inflammatory cytokine production in the NS1 pretreated cells compared to the control. Particularly, the pro-inflammatory mediators such as IL-6, CCL2 (MCP-1), TNFα, and CXCL10 transcripts were upregulated 50 fold or more with DV infection, and compared to HNS1 pretreated DCs, those receiving NS1 had a larger fold increase of these transcripts. CCL2, IL6, and TNFα are highly induced in sera of patients infected with DV (Tolfvenstam et al., 2011), and have been associated with vascular leakage and DHF (Huang et al., 2000, Lee et al., 2006, Stamatovic et al., 2005).

CCL2 is a monocyte chemoattractant that has been shown to recruit monocytes to the site of DV infection providing increased infection targets in the dermis (Schmid et al., 2014). CCL2 dependent monocyte recruitment has also been implicated in West Nile virus encephalitis pathogenesis, in which inflammatory Ly6Chi monocytes traffic to the brain of mice in a CCL2 dependent manner where they develop a microglial phenotype, and contribute to disease pathogenesis (Getts et al., 2008). Studies also provide evidence that TNFα and IL-6 contribute directly to endothelial cell permeability (Dewi et al., 2004, Maruo et al., 1992). If NS1 enhances pro-inflammatory cytokine production, this could lead to more recruitment of monocytes both as infection targets and pro-inflammatory mediators, which may contribute to dengue disease pathogenesis.

Chen et al. (Chen et al., 2015) recently reported that NS1 potentiates pro-inflammatory cytokine production during dengue infection of PBMCs. Two days post NS1 treatment, uninfected PBMCs upregulated TLR2 and TLR6 which led to production of IL-6 and TNFα. Upon DV infection, NS1 pretreatment also led to upregulation of these TLRs. In this model, TLR2 and TLR6 signaling decreased the survival of wild-type mice indicating a role for NS1 and these pattern recognition receptors (PRRs) in DV immunopathogenesis. Although we did not detect upregulation of IL-6 and TNFα with NS1 pretreatment alone, our treatment used a lower amount of NS1 for a shorter time period (20 μg/ml for 16 hours in our study, compared to 50 μg/ml for 48 hours), which may not have been enough time for induction of these pro-inflammatory mediators. Instead, we did observe upregulation of these cytokines during subsequent dengue infection. Our study describes a correlation between increased infectability of moDCs after NS1 pretreatment and increased cytokine production. One possibility is that simply increased infection would drive increased cytokine production. On the other hand we cannot exclude other mechanisms and direct effects of NS1 on cytokine production similar to some of the models described above.

Several studies indicate that NS1 may be a good vaccine target. Vaccination with NS1 adjuvanted with heat-labile toxin induced production of anti-NS1 antibodies, which decreased mortality of mice by 50%, and led to less exacerbated disease (Amorim et al., 2012). Another study showed that mice vaccinated with NS1, produced lower viremia in the serum, bone marrow, and spleen compared to mice vaccinated with OVA (Beatty et al., 2015). Taking these studies into consideration along with our findings that NS1 leads to enhanced dengue viral replication, it is possible that anti-NS1 antibodies could be playing a protective role by preventing NS1-dependent enhancement of infection, especially since the protected mice had lower serum viremia levels.

During human dengue infection, NS1 is secreted in the blood stream. Studies suggest a role for NS1 in complement activation, and a role for anti-NS1 antibodies in auto-reactivity and endothelial cell damage. However, there are no studies investigating uptake of NS1 by immunologically relevant cell types. In this report, we demonstrated that secreted DV1 NS1 associates with mo-DCs and enhances early viral RNA replication. This results in enhanced pro-inflammatory cytokine production during dengue infection. To our knowledge, this is the first study to directly investigate the effect of DV1 NS1 on DCs, the pivotal driver of immune responses and one of the main targets for DV infection in vivo. Our results are also important in light of the fact that many studies investigating the interaction of dengue viruses with DC and other target cell types use crude virus preparations that contain NS1 which as demonstrated here could significantly affect the interactions of the virus with its target cell.

Supplementary Material

1

Highlights.

DV1 NS1 protein is endocytosed by dendritic cells

Pretreatment of dendritic cells leads to enhanced infection by DV1

NS1 pretreated dendritic cells respond to DV1 with enhanced cytokine production

ACKNOWLEDGMENTS

This work was supported by a grant from the National Institutes of Health (NIH U54 AI 057157-07(SERCEB), project SE-RP-012.

Footnotes

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