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1 2 Altered immune response of immature dendritic cells upon dengue virus infection in the presence of specific antibodies 3 4 Silvia Torres1,2, Jacky Flipse1, Vinit C. Upasani1, Heidi van der Ende- Metselaar1, Silvio 5 Urcuqui-Inchima2, Jolanda M. Smit1, Izabela A. Rodenhuis-Zybert1 6 1Department 7 Center Groningen, Groningen, The Netherlands. 8 2Grupo 9 No. 52-21, Medellín, Colombia. of Medical Microbiology, University of Groningen and University Medical Inmunovirologia, Facultad de Medicina, Universidad de Antioquia UdeA, calle 70 10 Email addresses: 11 ST: [email protected] 12 JF: [email protected] 13 VU: [email protected] 14 SUI: [email protected], 15 HEM: [email protected], 16 JMS: [email protected], 17 IRZ: [email protected] 18 Corresponding author (Rodenhuis-Zybert, IA): Email: [email protected] 19 20 Word count: 2563 21 1 22 23 Abstract 24 Dengue virus (DENV) replication is known to prevent maturation of infected DCs thereby 25 impeding the development of adequate immunity. During secondary DENV infection, 26 dengue-specific antibodies can suppress DENV replication in immature DCs (immDCs), 27 however how dengue-antibody complexes (DENV-IC) influence DCs phenotype remains 28 elusive. Here, we evaluated the maturation state and cytokine profile of immDCs exposed 29 to DENV-ICs. Indeed, DENV infection of immDCs in the absence of antibodies was 30 hallmarked by blunted upregulation of CD83, CD86 and the major histocompatibility 31 complex molecule HLA-DR. In contrast, DENV infection in the presence of neutralizing 32 antibodies triggered full DCs maturation and induced a balanced inflammatory cytokine 33 response. Moreover, DENV infection at non-neutralizing conditions prompted upregulation 34 of CD83 and CD86 but not that of HLA-DR and triggered production of pro-inflammatory 35 cytokines. The effect of DENV-IC was found to rely on the engagement of FcγRIIa. 36 Altogether, our data show that the presence of DENV-IC alters the phenotype and cytokine 37 profile of DCs. 38 39 Introduction 40 Dengue disease is the most prevalent mosquito-borne viral disease with approximately 400 41 million infections per year worldwide (Bhatt et al., 2013). The disease is caused by 42 infection with any of the four serotypes of dengue virus (DENV1-4). Primary (1°) infections 43 are often asymptomatic and provide life-long protection against homotypic re-infection yet 44 short-term (1-3 years) protection against heterotypic re-infections (Reich et al., 2013). 45 Strikingly, after the cross-protective period, individuals re-infected with a heterologous 46 virus serotype are at risk of developing severe disease (Halstead, 2007). Severe disease is 47 also seen during 1° infections in infants with low titres of circulating maternal dengue 48 antibodies (Kliks et al., 1988; Simmons et al., 2007; Hause et al., 2015). Clearly, antibodies 49 play an important role in the outcome of DENV infection. Indeed, in vitro and in vivo studies 2 50 demonstrated that DENV infectivity depends on the concentrations and/or avidity of 51 antibodies present during infection. DENV infection is contained in the presence of 52 neutralizing antibody titers (de Alwis et al., 2011; Endy et al., 2004; van der Schaar et al., 53 2009). At non-neutralizing antibody concentrations however, enhanced infection of 54 immune cells (antibody-dependent enhancement (ADE) is seen due to viral entry via Fcγ 55 receptors (FcγR) (Rodenhuis-Zybert et al., 2010; Modhiran et al., 2010; Rodrigo et al., 2006; 56 Moi et al., 2010). 57 Dendritic cells (DCs) are the front line of defense against invading pathogens (Lutz & 58 Schuler, 2002). Upon antigen recognition, immature DCs (immDCs) acquire a mature 59 phenotype (matDCs) characterized by high surface expression of costimulatory molecules, 60 major histocompatibility complex II molecules (HLA-DR) and secretion of pro- and anti- 61 inflammatory cytokines (Banchereau & Steinman, 1998; Cella et al., 1997). Activation of 62 DCs is crucial for shaping the innate responses and initiation of adaptive immunity (Dudek 63 et al., 2013). However, DCs also represent an important target for DENV replication during 64 1° and 2° infections (Marovich et al., 2001; Wu et al., 2000; Schmid and Harris et al. 2014). 65 Indeed, in the absence of antibodies (direct infection), DENV not only efficiently infects 66 immDCs (Boonnak et al., 2008; Nightingale et al.,2008) but is also able to blunt their 67 maturation (Chang et al., 2012; Munoz-Jordan et al.,2003; Munoz-Jordan, 2010; Rodriguez- 68 Madozet al., 2010). High titers of DENV-specific antibodies are capable of neutralizing 69 DENV infection in immDCs (Boonnak et al., 2008). In the presence of non-neutralizing 70 antibody concentrations however, immDCs may support ADE of DENV infection (Boonnak 71 et al., 2008; Dejnirattisai et al., 2011 ). Remarkably, to date, little is known how DENV- 72 specific antibodies influence the activation of immDCs upon DENV infection. 73 In this study, we evaluated the effect of DENV-specific antibodies on immDC activation. 74 ImmDCs were generated from human monocytes isolated from buffy coats obtained from 75 healthy, anonymous volunteers (Sanquin Bloodbank) and processed in line with the 76 declaration of Helsinki. Briefly, human peripheral blood mononuclear cells (PBMC) were 77 isolated from buffy coats by centrifugation using Ficoll-Paque™ Plus (GE Healthcare). 78 Monocytes were isolated from PBMCs by gelatin adherence as previously described (Miller 79 et al., 2008) and allowed to differentiate into immDCs by culturing at 37oC in complete 3 80 RPMI medium (CM) with 20% fetal bovine serum (FBS), 500U/ml recombinant granulocyte 81 macrophage colony-stimulating factor (rGM-CSF), and 250U/ml recombinant interleukin-4 82 (IL-4; both from Prospec-Tany). Three-quarters of the culture medium was replaced every 83 second day for 6 days to generate immDCs. After 6 days of culture, the phenotype of the 84 cells was characterized as Lin-1neg, HLA-DRpos and CD11cpos (Supplementary Figure 1A), 85 thus confirming the differentiation of monocytes into immDCs (Boonnak et al., 2008). To 86 generate matDCs that would serve as a positive control in our experiments, immDCs were 87 stimulated at day 6 with MCM mimic containing 15 μg/ml recombinant IL-6, 500ng/ml 88 recombinant IL-1β, 500ng/ml TNF-α and 100μg/ml prostaglandin E2 (all from Bio 89 Connect), as described in Boonnak et al., 2008. At 24 hours post-stimulation, DCs showed 90 increased expression of CD40, CD83, CD86, HLA-DR compared to non-stimulated cells, 91 which confirms the inherent capacity of our immDCs to mature (Supplementary Figure 1B). 92 First, we analyzed the permissiveness of immDCs to DENV-2 infection. DENV-2 strain 93 16681 was propagated in Aedes albopictus cell line C6/36, as described before (Rodenhuis- 94 Zybert et al., 2010). The infectivity of DENV was determined by measuring the number of 95 plaque-forming units (PFU) by plaque assay (PA) on BHK-15 cells and the number of 96 genome-equivalent copies (GEc) by quantitative RT-PCR (qRT-PCR), as described 97 previously (van der Schaar et al., 2007; Zybert et al., 2008). The cells were infected with 98 DENV at multiplicity of infection (MOI) 0.1, 1, 10 at 37⁰C. At 2 hpi, the inoculum was 99 removed, cells were washed and cultured in fresh CM for another 22-24 hours. At 24- 100 26hpi, cells and supernatants were harvested and separated by low-speed centrifugation 101 (5’, 250g). The percentage of DENV-positive cells was evaluated by flow cytometry using 102 the DENV-2 envelope (E)-specific antibody 3H5 (Millipore) followed by a secondary anti- 103 mouse Alexa-647-labeled antibody (Figure 1A). As expected, the infection rate was found 104 to be MOI-dependent with the percentage of percentage of DENV E-positive varying 105 between the donors. Progeny virus production was measured in the cell-free supernatants 106 by plaque assay and qRT-PCR to obtain both PFU and GEc titers (Figure 1B). In agreement 107 with previous reports (Boonnak et al., 2008; Boonnak et al., 2011), immDCs are highly 108 permissive to DENV-2 replication. 4 109 To test the effect of antibodies on the permissiveness of immDCs to DENV-2, immDCs were 110 infected at MOI of 1 with DENV-2 alone (Direct infection) or DENV-2 immune-complexes 111 (DENV-IC). To generate DENV-IC, the virus was pre-incubated for 1 hour at 37°C with 10- 112 fold sequential dilutions of DENV-2-immune serum in a 1:1 volume ratio. In all 113 experiments, two distinct convalescent DENV-2 immune sera (kindly provided by G. 114 Comach Biomed-UC, Lardidev, Maracay, Venezuela; and T. Kochel, U.S. Naval Medical 115 Research Center Detachment, Lima, Peru) were used as a source of polyclonal antibodies. 116 The capacity of the sera to neutralize and enhance the DENV infection had been previously 117 confirmed in macrophage cell lines, human PBMCs and matDCs (Rodenhuis-Zybert et al., 118 2010 and unpublished). The DENV-immune sera used exhibited potent neutralizing activity 119 and inhibited DENV-2 infection at dilutions of 10-2 to 10-6 for three distinct donors 120 (Supplementary Figure 2, data for 10-3 dilution is shown in Figure 1C and 1D,). The PFU 121 and GEc titers were below the detection limit (20PFU/mL and 400 GEc/mL, respectively) 122 following infection with DENV pre-opsonized with 10-3 diluted serum (Figure 1D). 123 Therefore, we used this serum dilution for neutralization conditions (Neutr). As expected, 124 pre-incubation of DENV-2 with higher dilutions of the serum (Supplementary Figure 2; data 125 for 10- 8 dilution is shown in Figure 1) recovered DENV infectivity to the levels of direct 126 infection, as evidenced by flow cytometric analysis (Figure 1C) as well as DENV PFU and 127 GEc titers in the cell supernatants (Figure 1D). Hence, 10-8 serum dilution was used for non- 128 neutralizing conditions (Non-neutr) in subsequent experiments. Importantly, in line with a 129 previous report (Boonnak et al., 2008), none of the sera dilutions was able to enhance 130 DENV infection in immDCs (Supplementary Figure 2). 131 The effect of direct DENV infection on the maturation of immDCs has been studied 132 previously (Boonnak et al., 2008; Boonnak et al., 2001; Libraty et al., 2001; Navarro- 133 Sanchez et al., 2005; Nightingale et al., 2008; Palmer et al., 2005; Sun et al., 2009) yet the 134 effect of DENV-ICs on DCs remains unknown. To assess this, we next evaluated the surface 135 expression of the DCs maturation markers CD83, CD86 and HLA-DR 24hpi following 136 exposure of the cells to direct, UV-inactivated virus, non-neutralizing and neutralizing 137 DENV infection conditions. Twenty four hpi, cells were harvested, placed into cytometry 138 tubes and treated with FcRs blocking buffer (True Stain FcX, Biolegend) for 10 min at room 5 139 temperature. Subsequently, the cells were washed in staining medium [EDTA, saponin 140 (both from Sigma) and 2% FBS. Cell viability was evaluated using LIVE/DEAD® Fixable 141 Dead Cell Stain Kit (Invitrogen). Phenotyping of the cells with a panel of DCs markers and 142 intracellular DENV staining were performed, as described previously (Richter et al., 2014; 143 van der Schaar et al., 2008). Mock-infected (with or without the addition of neutralizing/ 144 non-neutralizing DENV-immune serum) and MCM mimic-stimulated cells (matDCs) were 145 added as negative and positive controls, respectively. 146 Figure 2 illustrates the effects of different infection conditions on the levels of CD83, CD86 147 and HLA-DR compared to matched mock infections. Expression of CD40 was upregulated 148 during all infection conditions (data not shown). In line with above-mentioned studies, 149 direct DENV infection did not promote significant upregulation of the maturation markers. 150 Exposure of cell to UV-inactivated DENV-2 (Direct UVi, complete inactivation confirmed by 151 PA) led to a modest increase in expression levels of CD83 and CD86, but had no effect on 152 HLA-DR. Interestingly, exposure of cells to DENV at neutralizing serum concentrations 153 triggered a significant increase in the expression of CD83, CD86 and HLA-DR when 154 compared to direct infection condition. In fact, the expression level of the maturation 155 markers found in DCs infected at neutralizing conditions was either higher (p>0.1 for 156 CD86) or comparable to that of matDCs (Figure 2). The observed upregulation was not 157 solely due to the blockage of DENV replication as expression of CD83 and HLA-DR 158 following exposure to UV-inactivated DENV where significantly lower (Figure 2). 159 Furthermore, DENV infection at non-neutralizing conditions also led to an increase in the 160 expression pattern of the maturation markers (with p<0.1 for CD86) as compared to direct 161 infection. Together, these results imply that the presence of DENV-IC promoted the 162 maturation capacity of DCs. 163 Subsequently, we examined whether differences in the expression level of DC markers 164 translated to a particular cytokine expression profile. To this end, we evaluated the levels 165 of pro- (IL-6, TNF-α), and anti-inflammatory cytokines (IL-10, IL-4) in the supernatants of 166 DCs infected at conditions as described above (Figure 3A). In line with the lack of DCs 167 maturation, direct infection did not significantly alter the levels of the aforementioned 168 cytokines when compared to mock infection. Importantly, when immDCs were exposed to 6 169 neutralizing DENV-IC, the cells secreted significantly higher amounts of IL-6, TNF-α, IL-4 170 and IL-10 when compared to direct infection. In line with the data obtained for surface 171 markers, addition of the neutralizing serum alone (Figure 3B), as well as Direct UVi (data 172 not shown), did not trigger significant changes in the cytokine response. Taken together, 173 the observed increase upon infection in presence of the serum was likely due to the 174 presence of DENV-IC. This notion was further supported by the observation that non- 175 neutralizing DENV-infection conditions resulted in higher, albeit variable between the 176 donors, levels of IL-6, IL-4 and TNF-α than those induced by the direct infection (Figure 177 3A). Interestingly, it has recently been reported that induction of pro-inflammatory 178 cytokines following immune-complex stimulation depends mainly on FcγRIIa (Vogelpoel 179 L.T.C. et al., 2014). Ligation of FcγRIIa is also required for the production of IL-6 and TNF-α 180 following infection of matDCs under ADE conditions (Boonnak et al., 2008). To assess the 181 role of FcγRIIa in the activation of immDCs in the context of DENV-infection, we pre-treated 182 the immDCs with for 30 minutes with 5mg/mL of FcγRIIa-blocking antibody, clone IV.3 183 (Stem Cell). Figure 3B and 3C show that blocking FcγRIIa had no effect on the levels of 184 cytokines in the conditions of mock infections. However, it did prevent the production of 185 cytokines during infection in the presence of neutralizing (Figure 3B) and non-neutralizing 186 (Figure 3C) serum concentrations. Thus, FcγRIIa ligation was responsible for the 187 production of IL-6 and TNF-α following infection with DENV-IC. Of note, blocking of FcγRIIa 188 had no effect on the neutralizing capacity of the antibodies or on the level of infection at 189 non-neutralizing conditions (Supplementary Figure S3). 190 In summary, our data demonstrate that the presence of DENV-IC triggers distinct DC 191 phenotypes and cytokine profiles. 192 The main role of DCs is to sense, process and present antigens of invading pathogens to 193 cells of the adaptive immune system (Banchereau & Steinman, 1998). Viruses as HIV-1, 194 measles virus, vaccinia virus and DENV target DCs for replication (Boonnak et al., 2008; de 195 Witte et al.,2006; Dejnirattisai et al., 2011; Ho et al., 2001; Liu et al., 2008; Marovich et al., 196 2001; Nightingale et al., 2008; Rinaldo, 2013; Wu et al., 2000). Given the pivotal role of DCs 197 in promoting adaptive immune responses, it is not surprising that many viruses impair the 198 ability of infected DCs to initiate adaptive immunity (Lilley & Ploegh, 2005; Oreshkova et 7 199 al., 2015). Indeed, several studies have shown that DC maturation is blunted upon DENV 200 infection (Chang et al., 2012; Munoz-Jordan et al., 2003; Rodriguez-Madoz et al., 2010; 201 Palmer et al., 2005). In agreement with this, we found that DENV replication impedes DCs 202 maturation. Although not tested here, previous study has shown that DENV infection in DCs 203 impairs their antigen-presenting cell function (Palmer et al., 2005). Indeed, clinical data 204 show that antigen-presenting cells in patients suffering from acute DV infections are unable 205 to stimulate T-cell responses to mitogens and DV antigens (Mathew, A., et al, 1999). Our 206 data suggest that the presence of DENV-specific antibodies may exert distinct 207 immunomodulatory effects in immDCs during 2° infection. At conditions of antibody- 208 mediated virus neutralization, the expression of HLA-DR, CD83 and CD86 is up-regulated to 209 levels similar as mock-infected matDCs. Indeed, binding of ICs to FcRs on DCs is known to 210 trigger phagocytosis, presentation of antigenic peptides on MHC class I and class II 211 molecules and, depending on the FcR, differential cytokine production (den Dunnen et al., 212 2012; Nimmerjahn & Ravetch, 2008; Vogelpoel L.T.C. et al., 2014). In line with this, we 213 observed increased production of pro-inflammatory (IL-6, and TNF-α) and anti- 214 inflammatory (IL-4 and IL-10) cytokines at conditions of DENV neutralization. 215 Exposure of immDCs to DENV-ICs at non-neutralizing conditions triggered a significant 216 increase of CD83 and CD86 but did not alter the expression patterns of the HLA-DR. 217 Moreover, TNF-α, IL-6, and IL-4 but not IL-10 were released at this condition. The presence 218 of IL-4 is known to inhibit IL-10 production by DCs (Yao et al., 2005). It is possible that the 219 lack of IL-10 production is due to the significantly elevated levels of IL-4 released from DCs 220 exposed to non-neutralizing conditions. Notably, balanced levels of TNF-α and IL-10 have 221 been shown to be important for control the inflammatory responses (van der Poll et al., 222 1995, Gudmundsson et al., 1998). Thus it is tempting to speculate that this might be yet 223 another mechanism contributing to a cytokine storm observed in course of severe dengue 224 (Costa et al., 2013; Pang et al., 2007; Soundravally et al., 2013). Mechanistically, the overall 225 increase in cytokine production upon infection with non-neutralized DENV-ICs compared 226 to direct infection suggests that FcRs-mediated infection triggers different downstream 227 signaling pathways. 8 228 The presence of DENV-specific antibodies during DENV infection of DCs has consequences 229 for the maturation of immDCs and subsequent cytokine responses. Our results corroborate 230 earlier findings that DENV-2 can blunt the maturation and activation of exposed DCs. 231 Importantly, we show that the presence of high concentrations of DENV-specific antibodies 232 does not only neutralize DENV infection of immDCs but also rescues the ability of immDCs 233 to mature and produce pro- and anti-inflammatory cytokines. On the other hand, infection 234 in the presence of non-neutralizing antibody titers may induce a phenotype of DCs with 235 reduced ability to present antigens while triggering mainly pro-inflammatory responses. 236 Our data also showed that the ability of DCs to acquire these distinct phenotypes relies on 237 FcγRIIa ligation. Further studies should investigate whether this partially impaired DCs 238 phenotype contributes to the aberrant T responses and exacerbation of inflammation seen 239 during severe disease (Green & Rothman, 2006; Mangada & Rothman, 2005; Rothman, 240 2011). 241 242 Acknowledgments 243 JF and JMS were supported by Dutch Scientific Organization (NWO) VIDI-grant to JMS. ST 244 was supported by Colciencias, Colombia (#111551928777) and SUI by Universidad de 245 Antioquia, (Programa de Sostenibilidad 2016-2017) and UMCG. IRZ was supported by 246 NWO VENI-grant to IRZ. 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The 462 Journal of General Virology, 89(Pt 12), 3047-3051. doi:10.1099/vir.0.2008/002535-0; 463 10.1099/vir.0.2008/002535-0 in Living virus Cells. infection. PLoS Pathogens, Immunobiology, 464 17 4(12), 214(7), e1000244. 613-629. Nat. Commun. 5:5444 doi: 465 Figure legends 466 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