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Altered immune response of immature dendritic cells upon dengue virus
infection in the presence of specific antibodies
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Silvia Torres1,2, Jacky Flipse1, Vinit C. Upasani1, Heidi van der Ende- Metselaar1, Silvio
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Urcuqui-Inchima2, Jolanda M. Smit1, Izabela A. Rodenhuis-Zybert1
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1Department
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Center Groningen, Groningen, The Netherlands.
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2Grupo
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No. 52-21, Medellín, Colombia.
of Medical Microbiology, University of Groningen and University Medical
Inmunovirologia, Facultad de Medicina, Universidad de Antioquia UdeA, calle 70
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Email addresses:
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ST: [email protected]
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JF: [email protected]
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VU: [email protected]
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SUI: [email protected],
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HEM: [email protected],
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JMS: [email protected],
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IRZ: [email protected]
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Corresponding author (Rodenhuis-Zybert, IA): Email: [email protected]
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Word count: 2563
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Abstract
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Dengue virus (DENV) replication is known to prevent maturation of infected DCs thereby
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impeding the development of adequate immunity. During secondary DENV infection,
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dengue-specific antibodies can suppress DENV replication in immature DCs (immDCs),
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however how dengue-antibody complexes (DENV-IC) influence DCs phenotype remains
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elusive. Here, we evaluated the maturation state and cytokine profile of immDCs exposed
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to DENV-ICs. Indeed, DENV infection of immDCs in the absence of antibodies was
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hallmarked by blunted upregulation of CD83, CD86 and the major histocompatibility
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complex molecule HLA-DR. In contrast, DENV infection in the presence of neutralizing
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antibodies triggered full DCs maturation and induced a balanced inflammatory cytokine
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response. Moreover, DENV infection at non-neutralizing conditions prompted upregulation
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of CD83 and CD86 but not that of HLA-DR and triggered production of pro-inflammatory
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cytokines. The effect of DENV-IC was found to rely on the engagement of FcγRIIa.
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Altogether, our data show that the presence of DENV-IC alters the phenotype and cytokine
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profile of DCs.
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Introduction
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Dengue disease is the most prevalent mosquito-borne viral disease with approximately 400
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million infections per year worldwide (Bhatt et al., 2013). The disease is caused by
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infection with any of the four serotypes of dengue virus (DENV1-4). Primary (1°) infections
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are often asymptomatic and provide life-long protection against homotypic re-infection yet
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short-term (1-3 years) protection against heterotypic re-infections (Reich et al., 2013).
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Strikingly, after the cross-protective period, individuals re-infected with a heterologous
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virus serotype are at risk of developing severe disease (Halstead, 2007). Severe disease is
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also seen during 1° infections in infants with low titres of circulating maternal dengue
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antibodies (Kliks et al., 1988; Simmons et al., 2007; Hause et al., 2015). Clearly, antibodies
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play an important role in the outcome of DENV infection. Indeed, in vitro and in vivo studies
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demonstrated that DENV infectivity depends on the concentrations and/or avidity of
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antibodies present during infection. DENV infection is contained in the presence of
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neutralizing antibody titers (de Alwis et al., 2011; Endy et al., 2004; van der Schaar et al.,
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2009). At non-neutralizing antibody concentrations however, enhanced infection of
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immune cells (antibody-dependent enhancement (ADE) is seen due to viral entry via Fcγ
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receptors (FcγR) (Rodenhuis-Zybert et al., 2010; Modhiran et al., 2010; Rodrigo et al., 2006;
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Moi et al., 2010).
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Dendritic cells (DCs) are the front line of defense against invading pathogens (Lutz &
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Schuler, 2002). Upon antigen recognition, immature DCs (immDCs) acquire a mature
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phenotype (matDCs) characterized by high surface expression of costimulatory molecules,
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major histocompatibility complex II molecules (HLA-DR) and secretion of pro- and anti-
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inflammatory cytokines (Banchereau & Steinman, 1998; Cella et al., 1997). Activation of
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DCs is crucial for shaping the innate responses and initiation of adaptive immunity (Dudek
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et al., 2013). However, DCs also represent an important target for DENV replication during
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1° and 2° infections (Marovich et al., 2001; Wu et al., 2000; Schmid and Harris et al. 2014).
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Indeed, in the absence of antibodies (direct infection), DENV not only efficiently infects
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immDCs (Boonnak et al., 2008; Nightingale et al.,2008) but is also able to blunt their
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maturation (Chang et al., 2012; Munoz-Jordan et al.,2003; Munoz-Jordan, 2010; Rodriguez-
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Madozet al., 2010). High titers of DENV-specific antibodies are capable of neutralizing
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DENV infection in immDCs (Boonnak et al., 2008). In the presence of non-neutralizing
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antibody concentrations however, immDCs may support ADE of DENV infection (Boonnak
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et al., 2008; Dejnirattisai et al., 2011 ). Remarkably, to date, little is known how DENV-
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specific antibodies influence the activation of immDCs upon DENV infection.
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In this study, we evaluated the effect of DENV-specific antibodies on immDC activation.
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ImmDCs were generated from human monocytes isolated from buffy coats obtained from
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healthy, anonymous volunteers (Sanquin Bloodbank) and processed in line with the
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declaration of Helsinki. Briefly, human peripheral blood mononuclear cells (PBMC) were
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isolated from buffy coats by centrifugation using Ficoll-Paque™ Plus (GE Healthcare).
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Monocytes were isolated from PBMCs by gelatin adherence as previously described (Miller
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et al., 2008) and allowed to differentiate into immDCs by culturing at 37oC in complete
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RPMI medium (CM) with 20% fetal bovine serum (FBS), 500U/ml recombinant granulocyte
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macrophage colony-stimulating factor (rGM-CSF), and 250U/ml recombinant interleukin-4
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(IL-4; both from Prospec-Tany). Three-quarters of the culture medium was replaced every
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second day for 6 days to generate immDCs. After 6 days of culture, the phenotype of the
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cells was characterized as Lin-1neg, HLA-DRpos and CD11cpos (Supplementary Figure 1A),
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thus confirming the differentiation of monocytes into immDCs (Boonnak et al., 2008). To
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generate matDCs that would serve as a positive control in our experiments, immDCs were
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stimulated at day 6 with MCM mimic containing 15 μg/ml recombinant IL-6, 500ng/ml
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recombinant IL-1β, 500ng/ml TNF-α and 100μg/ml prostaglandin E2 (all from Bio
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Connect), as described in Boonnak et al., 2008. At 24 hours post-stimulation, DCs showed
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increased expression of CD40, CD83, CD86, HLA-DR compared to non-stimulated cells,
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which confirms the inherent capacity of our immDCs to mature (Supplementary Figure 1B).
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First, we analyzed the permissiveness of immDCs to DENV-2 infection. DENV-2 strain
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16681 was propagated in Aedes albopictus cell line C6/36, as described before (Rodenhuis-
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Zybert et al., 2010). The infectivity of DENV was determined by measuring the number of
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plaque-forming units (PFU) by plaque assay (PA) on BHK-15 cells and the number of
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genome-equivalent copies (GEc) by quantitative RT-PCR (qRT-PCR), as described
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previously (van der Schaar et al., 2007; Zybert et al., 2008). The cells were infected with
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DENV at multiplicity of infection (MOI) 0.1, 1, 10 at 37⁰C. At 2 hpi, the inoculum was
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removed, cells were washed and cultured in fresh CM for another 22-24 hours. At 24-
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26hpi, cells and supernatants were harvested and separated by low-speed centrifugation
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(5’, 250g). The percentage of DENV-positive cells was evaluated by flow cytometry using
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the DENV-2 envelope (E)-specific antibody 3H5 (Millipore) followed by a secondary anti-
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mouse Alexa-647-labeled antibody (Figure 1A). As expected, the infection rate was found
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to be MOI-dependent with the percentage of percentage of DENV E-positive varying
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between the donors. Progeny virus production was measured in the cell-free supernatants
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by plaque assay and qRT-PCR to obtain both PFU and GEc titers (Figure 1B). In agreement
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with previous reports (Boonnak et al., 2008; Boonnak et al., 2011), immDCs are highly
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permissive to DENV-2 replication.
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To test the effect of antibodies on the permissiveness of immDCs to DENV-2, immDCs were
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infected at MOI of 1 with DENV-2 alone (Direct infection) or DENV-2 immune-complexes
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(DENV-IC). To generate DENV-IC, the virus was pre-incubated for 1 hour at 37°C with 10-
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fold sequential dilutions of DENV-2-immune serum in a 1:1 volume ratio. In all
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experiments, two distinct convalescent DENV-2 immune sera (kindly provided by G.
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Comach Biomed-UC, Lardidev, Maracay, Venezuela; and T. Kochel, U.S. Naval Medical
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Research Center Detachment, Lima, Peru) were used as a source of polyclonal antibodies.
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The capacity of the sera to neutralize and enhance the DENV infection had been previously
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confirmed in macrophage cell lines, human PBMCs and matDCs (Rodenhuis-Zybert et al.,
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2010 and unpublished). The DENV-immune sera used exhibited potent neutralizing activity
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and inhibited DENV-2 infection at dilutions of 10-2 to 10-6 for three distinct donors
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(Supplementary Figure 2, data for 10-3 dilution is shown in Figure 1C and 1D,). The PFU
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and GEc titers were below the detection limit (20PFU/mL and 400 GEc/mL, respectively)
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following infection with DENV pre-opsonized with 10-3 diluted serum (Figure 1D).
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Therefore, we used this serum dilution for neutralization conditions (Neutr). As expected,
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pre-incubation of DENV-2 with higher dilutions of the serum (Supplementary Figure 2; data
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for 10- 8 dilution is shown in Figure 1) recovered DENV infectivity to the levels of direct
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infection, as evidenced by flow cytometric analysis (Figure 1C) as well as DENV PFU and
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GEc titers in the cell supernatants (Figure 1D). Hence, 10-8 serum dilution was used for non-
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neutralizing conditions (Non-neutr) in subsequent experiments. Importantly, in line with a
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previous report (Boonnak et al., 2008), none of the sera dilutions was able to enhance
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DENV infection in immDCs (Supplementary Figure 2).
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The effect of direct DENV infection on the maturation of immDCs has been studied
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previously (Boonnak et al., 2008; Boonnak et al., 2001; Libraty et al., 2001; Navarro-
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Sanchez et al., 2005; Nightingale et al., 2008; Palmer et al., 2005; Sun et al., 2009) yet the
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effect of DENV-ICs on DCs remains unknown. To assess this, we next evaluated the surface
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expression of the DCs maturation markers CD83, CD86 and HLA-DR 24hpi following
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exposure of the cells to direct, UV-inactivated virus, non-neutralizing and neutralizing
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DENV infection conditions. Twenty four hpi, cells were harvested, placed into cytometry
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tubes and treated with FcRs blocking buffer (True Stain FcX, Biolegend) for 10 min at room
5
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temperature. Subsequently, the cells were washed in staining medium [EDTA, saponin
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(both from Sigma) and 2% FBS. Cell viability was evaluated using LIVE/DEAD® Fixable
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Dead Cell Stain Kit (Invitrogen). Phenotyping of the cells with a panel of DCs markers and
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intracellular DENV staining were performed, as described previously (Richter et al., 2014;
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van der Schaar et al., 2008). Mock-infected (with or without the addition of neutralizing/
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non-neutralizing DENV-immune serum) and MCM mimic-stimulated cells (matDCs) were
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added as negative and positive controls, respectively.
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Figure 2 illustrates the effects of different infection conditions on the levels of CD83, CD86
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and HLA-DR compared to matched mock infections. Expression of CD40 was upregulated
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during all infection conditions (data not shown). In line with above-mentioned studies,
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direct DENV infection did not promote significant upregulation of the maturation markers.
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Exposure of cell to UV-inactivated DENV-2 (Direct UVi, complete inactivation confirmed by
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PA) led to a modest increase in expression levels of CD83 and CD86, but had no effect on
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HLA-DR. Interestingly, exposure of cells to DENV at neutralizing serum concentrations
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triggered a significant increase in the expression of CD83, CD86 and HLA-DR when
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compared to direct infection condition. In fact, the expression level of the maturation
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markers found in DCs infected at neutralizing conditions was either higher (p>0.1 for
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CD86) or comparable to that of matDCs (Figure 2). The observed upregulation was not
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solely due to the blockage of DENV replication as expression of CD83 and HLA-DR
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following exposure to UV-inactivated DENV where significantly lower (Figure 2).
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Furthermore, DENV infection at non-neutralizing conditions also led to an increase in the
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expression pattern of the maturation markers (with p<0.1 for CD86) as compared to direct
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infection. Together, these results imply that the presence of DENV-IC promoted the
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maturation capacity of DCs.
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Subsequently, we examined whether differences in the expression level of DC markers
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translated to a particular cytokine expression profile. To this end, we evaluated the levels
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of pro- (IL-6, TNF-α), and anti-inflammatory cytokines (IL-10, IL-4) in the supernatants of
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DCs infected at conditions as described above (Figure 3A). In line with the lack of DCs
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maturation, direct infection did not significantly alter the levels of the aforementioned
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cytokines when compared to mock infection. Importantly, when immDCs were exposed to
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neutralizing DENV-IC, the cells secreted significantly higher amounts of IL-6, TNF-α, IL-4
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and IL-10 when compared to direct infection. In line with the data obtained for surface
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markers, addition of the neutralizing serum alone (Figure 3B), as well as Direct UVi (data
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not shown), did not trigger significant changes in the cytokine response. Taken together,
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the observed increase upon infection in presence of the serum was likely due to the
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presence of DENV-IC. This notion was further supported by the observation that non-
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neutralizing DENV-infection conditions resulted in higher, albeit variable between the
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donors, levels of IL-6, IL-4 and TNF-α than those induced by the direct infection (Figure
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3A). Interestingly, it has recently been reported that induction of pro-inflammatory
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cytokines following immune-complex stimulation depends mainly on FcγRIIa (Vogelpoel
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L.T.C. et al., 2014). Ligation of FcγRIIa is also required for the production of IL-6 and TNF-α
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following infection of matDCs under ADE conditions (Boonnak et al., 2008). To assess the
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role of FcγRIIa in the activation of immDCs in the context of DENV-infection, we pre-treated
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the immDCs with for 30 minutes with 5mg/mL of FcγRIIa-blocking antibody, clone IV.3
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(Stem Cell). Figure 3B and 3C show that blocking FcγRIIa had no effect on the levels of
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cytokines in the conditions of mock infections. However, it did prevent the production of
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cytokines during infection in the presence of neutralizing (Figure 3B) and non-neutralizing
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(Figure 3C) serum concentrations. Thus, FcγRIIa ligation was responsible for the
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production of IL-6 and TNF-α following infection with DENV-IC. Of note, blocking of FcγRIIa
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had no effect on the neutralizing capacity of the antibodies or on the level of infection at
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non-neutralizing conditions (Supplementary Figure S3).
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In summary, our data demonstrate that the presence of DENV-IC triggers distinct DC
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phenotypes and cytokine profiles.
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The main role of DCs is to sense, process and present antigens of invading pathogens to
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cells of the adaptive immune system (Banchereau & Steinman, 1998). Viruses as HIV-1,
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measles virus, vaccinia virus and DENV target DCs for replication (Boonnak et al., 2008; de
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Witte et al.,2006; Dejnirattisai et al., 2011; Ho et al., 2001; Liu et al., 2008; Marovich et al.,
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2001; Nightingale et al., 2008; Rinaldo, 2013; Wu et al., 2000). Given the pivotal role of DCs
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in promoting adaptive immune responses, it is not surprising that many viruses impair the
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ability of infected DCs to initiate adaptive immunity (Lilley & Ploegh, 2005; Oreshkova et
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al., 2015). Indeed, several studies have shown that DC maturation is blunted upon DENV
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infection (Chang et al., 2012; Munoz-Jordan et al., 2003; Rodriguez-Madoz et al., 2010;
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Palmer et al., 2005). In agreement with this, we found that DENV replication impedes DCs
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maturation. Although not tested here, previous study has shown that DENV infection in DCs
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impairs their antigen-presenting cell function (Palmer et al., 2005). Indeed, clinical data
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show that antigen-presenting cells in patients suffering from acute DV infections are unable
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to stimulate T-cell responses to mitogens and DV antigens (Mathew, A., et al, 1999). Our
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data suggest that the presence of DENV-specific antibodies may exert distinct
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immunomodulatory effects in immDCs during 2° infection. At conditions of antibody-
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mediated virus neutralization, the expression of HLA-DR, CD83 and CD86 is up-regulated to
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levels similar as mock-infected matDCs. Indeed, binding of ICs to FcRs on DCs is known to
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trigger phagocytosis, presentation of antigenic peptides on MHC class I and class II
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molecules and, depending on the FcR, differential cytokine production (den Dunnen et al.,
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2012; Nimmerjahn & Ravetch, 2008; Vogelpoel L.T.C. et al., 2014). In line with this, we
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observed increased production of pro-inflammatory (IL-6, and TNF-α) and anti-
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inflammatory (IL-4 and IL-10) cytokines at conditions of DENV neutralization.
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Exposure of immDCs to DENV-ICs at non-neutralizing conditions triggered a significant
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increase of CD83 and CD86 but did not alter the expression patterns of the HLA-DR.
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Moreover, TNF-α, IL-6, and IL-4 but not IL-10 were released at this condition. The presence
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of IL-4 is known to inhibit IL-10 production by DCs (Yao et al., 2005). It is possible that the
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lack of IL-10 production is due to the significantly elevated levels of IL-4 released from DCs
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exposed to non-neutralizing conditions. Notably, balanced levels of TNF-α and IL-10 have
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been shown to be important for control the inflammatory responses (van der Poll et al.,
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1995, Gudmundsson et al., 1998). Thus it is tempting to speculate that this might be yet
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another mechanism contributing to a cytokine storm observed in course of severe dengue
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(Costa et al., 2013; Pang et al., 2007; Soundravally et al., 2013). Mechanistically, the overall
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increase in cytokine production upon infection with non-neutralized DENV-ICs compared
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to direct infection suggests that FcRs-mediated infection triggers different downstream
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signaling pathways.
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The presence of DENV-specific antibodies during DENV infection of DCs has consequences
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for the maturation of immDCs and subsequent cytokine responses. Our results corroborate
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earlier findings that DENV-2 can blunt the maturation and activation of exposed DCs.
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Importantly, we show that the presence of high concentrations of DENV-specific antibodies
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does not only neutralize DENV infection of immDCs but also rescues the ability of immDCs
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to mature and produce pro- and anti-inflammatory cytokines. On the other hand, infection
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in the presence of non-neutralizing antibody titers may induce a phenotype of DCs with
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reduced ability to present antigens while triggering mainly pro-inflammatory responses.
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Our data also showed that the ability of DCs to acquire these distinct phenotypes relies on
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FcγRIIa ligation. Further studies should investigate whether this partially impaired DCs
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phenotype contributes to the aberrant T responses and exacerbation of inflammation seen
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during severe disease (Green & Rothman, 2006; Mangada & Rothman, 2005; Rothman,
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2011).
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Acknowledgments
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JF and JMS were supported by Dutch Scientific Organization (NWO) VIDI-grant to JMS. ST
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was supported by Colciencias, Colombia (#111551928777) and SUI by Universidad de
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Antioquia, (Programa de Sostenibilidad 2016-2017) and UMCG. IRZ was supported by
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NWO VENI-grant to IRZ. The funders had no role in study design, data collection and
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analysis, decision to publish, or preparation of the manuscript.
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Author Contributions
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Conceived and designed the experiments: ST, JF, VU, IRZ
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Performed the experiments: ST, JF, VU, HEM
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Analyzed the data: ST, JF, VU, IRZ, JMS, SUI
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Contributed with reagents/materials/analysis tools: SUI and JMS
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Wrote the paper: ST, VU, JMS and IRZ
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Figure legends
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Figure 1. DENV infection of immDCs in the absence or presence of DENV-immune
467
serum. ImmDCs were infected with DENV-2 at MOI 0.1, 1 or 10. Cells and cell supernatants
468
were harvested at 24 hours post-infection (hpi). (A) The percentage of DENV-positive cells
469
was determined using mAb 3H5. (B) Quantitative analysis of the infectious properties of
470
DENV in immDCs. White bars: number of infectious particles (Log10 PFU/ml); black bars:
471
number of genome-equivalent copies (log10 GEc/mL). Results are representative of 10
472
independent experiments with 3 donors. (C) ImmDCs were infected either in the absence of
473
DENV immune serum, under neutralizing (here represented by 10-3) or non-neutralizing
474
conditions (here represented by 10-8). (D) Quantitative analysis of the infectious properties
475
of DENV in immDCs under various infection conditions. White bars: number of infectious
476
particles (Log10 PFU/ml); black bars: number of genome-equivalent copies (log10 GEc/mL).
477
Results are representative of ≥10 experiments with three donors. Bars represent the
478
standard error of the mean (SEM), n.d. denotes not detected.
479
480
Figure 2. Differences in DC phenotype following direct DENV infection and infection
481
in the presence of antibodies. ImmDCs were infected with C6/36-derived virus or UV-
482
inactivated (UVi) DENV at MOI 1 in the absence or presence of DENV-immune serum. The
483
mean fluorescence intensity (MFI) of the expression of co-stimulatory markers CD83 and
484
CD86 and the major histocompatibility complex molecule HLA-DR was assessed by flow
485
cytometry at 24 hpi. The bars represent fold-changes between different infection
486
conditions and their matched mocks obtained from at least 3 independent experiments ±
487
SEM. Statistical analysis was done by use of Mann-Whitney U-test (*P< 0.05 ** P < 0.01).
488
Stars above the bars indicate differences when compared to mock-infected cells.
489
490
Figure 3. DENV-immune complexes stimulate cytokine secretion by DCs in a FcγRIIa-
491
dependent manner. DENV infection (MOI 1) was performed as described in the legend to
492
Figure 2. (A) IL-6, TNF-α, IL-10 and IL-4 production was measured by a Cytokine Bead
493
Assay (CBA, BD Biosciences). (B & C) The effect of anti-FcγRIIa antibody pre-treatment on
494
the levels of pro-inflammatory cytokines produced by immDCs following (B) neutralizing
18
495
and (C) non-neutralizing infection conditions. The data shown are representative of at least
496
3 independent experiments ±SD. Statistical analysis was done by use of Mann-Whitney U-
497
test (* P< 0.05; ** P < 0.01, *** P <0.001). Stars above the bars indicate differences when
498
compared to mock-infected cells.
499
500
Suplementary data
501
Supplementary Figure 1. Phenotypic analysis of immDCs and matDCs. (A) Phenotypic
502
analysis of monocyte-derived immDCs. A total of 1.5x105 cells were counted. Histograms
503
show the fluorescence intensity of typical dendritic cell markers: Lin-1, HLA-DR and CD11c.
504
Dashed line: isotype control; continuous line: specific antibody. (B) MFI of the co-
505
stimulatory markers CD40, CD83, and CD86 of immDCs and matDCs.
506
507
Supplementary Figure 2. Effect of the dengue- immune sera dilution on DENV-2
508
infection of immDCs. Infection was performed as described in the legend to Figure 1. Cell-
509
free-supernatants of 24hpi were analyzed by plaque assay. The data shown are
510
representative of two independent experiments with three donors.
511
512
513
Supplementary Figure 3. Effect of FcγRIIa blockage on DENV infection in immDCs in
514
the presence of neutralizing and non-neutralizing sera DENV infection was performed
515
as described in the legend to Figure 3. Cell-free-supernatants of 24hpi were analyzed by
516
plaque assay. The data shown are representative of three independent experiments.
517
19