Original Paper Intervirology 2015;58:250–259 DOI: 10.1159/000440723

Received: May 20, 2015 Accepted after revision: August 27, 2015 Published online: October 24, 2015

Dengue Virus Serotype-2 Interferes with the Formation of Neutrophil Extracellular Traps Maria Maximina B. Moreno-Altamirano a Oscar Rodríguez-Espinosa a Oscar Rojas-Espinosa b Bernardo Pliego-Rivero c Francisco J. Sánchez-García a Laboratorios de a Inmunorregulación and b Inmunobiología, Departamento de Inmunología, Escuela Nacional de Ciencias Biológicas, Instituto Politécnico Nacional, and c Universidad Autónoma del Estado de México, Mexico City, Mexico

Abstract Objectives: Neutrophils play an important role in the control of pathogens through several mechanisms, including phagocytosis and the formation of neutrophil extracellular traps (NETs). The latter consists of DNA as a backbone with embedded antimicrobial peptides, histones, and proteases, providing a matrix to entrap and in some cases to kill microbes. Some metabolic requirements for NET formation have recently been described. The virus-induced formation of NETs and the role of these traps in viral infections remain scarcely reported. Here, we analyzed whether dengue virus serotype-2 (DENV-2) induces NET formation and the DENV-2 effect on phorbol myristate acetate (PMA)-induced NETs. Methods: Peripheral blood-derived neutrophils were exposed in vitro to DENV-2 or exposed to DENV-2 and then stimulated with PMA. NET formation was assessed by fluorescence microscopy. Cell membrane Glut-1, glucose uptake, and reactive oxygen species (ROS) production were assessed. Results: DENV-2 does not induce the formation of NETs. Moreover, DENV-2 inhibits PMA-induced formation of

© 2015 S. Karger AG, Basel 0300–5526/15/0584–0250$39.50/0 E-Mail [email protected] www.karger.com/int

NETs by about 80%. This effect is not related to the production of ROS. The mechanism seemingly accountable for this inhibitory effect is the DENV-2-mediated inhibition of PMAinduced glucose uptake by neutrophils. Conclusion: Our results suggest that DENV-2 inhibits glucose uptake as a metabolism-based way to avoid the formation of NETs. © 2015 S. Karger AG, Basel

Introduction

Neutrophils are short-lived cells of the innate immune system with the ability to phagocytize and kill microorganisms [1]. Upon activation, they also release neutrophil extracellular traps (NETs) made up of chromatin, either nuclear (from dead or dying cells) or mitochondrial (from living cells), that in some instances have microbicidal activity [2–6]. A growing list of microorganisms has been shown to stimulate the release of NETs, among which are a few viruses [7]. In addition to lymphocytes, natural killer cells, and antigen-presenting cells (regarded as the main effector cells in viral infections), there is also a role for early inMaría Maximina B. Moreno-Altamirano, PhD Laboratorio de Inmunorregulación, Departamento de Inmunología Escuela Nacional de Ciencias Biológicas, Instituto Politécnico Nacional Carpio y Plan de Ayala, Col Santo Tomás, Mexico City CP 11340 (Mexico) E-Mail bertha.moreno.altamirano @ gmail.com

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Key Words Dengue virus · Neutrophils · Neutrophil extracellular traps

DENV-2-Mediated Inhibition of NETs

Materials and Methods Dengue Virus DENV-2 New Guinea strain was obtained from Aedes albopictus C6/36-infected cells. When the cell culture reached about 80% of confluence, the DENV was purified and kept frozen at –70° until use. Neutrophil Isolation and in vitro Induction of NETs Neutrophils were isolated from 5–10 ml of peripheral blood of healthy donors (average age 23 years), both females and males (gender had no influence on the results), using heparin as anticoagulant and gradient centrifugation on PolymorphprepTM (AxisShield, Oslo, Norway; volume/volume, 300 g for 50 min at 4°). The polymorph nuclear cell-containing interface was recovered and washed twice with PBS pH 7.2 (300 g for 5 min at 4°). Neutrophils were suspended in RPMI culture medium (in vitro), supplemented with 2% FBS (Gibco; Invitrogen, Grand Island, N.Y., USA) and used immediately. The institutional (ENCB-IPN) bioethics committee reviewed this protocol. For the formation of NETs, neutrophils (3 × 105) were seeded in duplicate on 8-well Lab-Tek tissue culture chambers (Thermo Fisher Scientific, Inc., Waltham, Mass., USA) in RPMI culture medium. The neutrophils were stimulated with 100 ng/ml PMA and incubated at 37° in a 5% CO2 atmosphere for 3 h and then fixed with 4% paraformaldehyde for 20 min, washed twice with PBS, stained with DAPI (Thermo Fisher Scientific, Inc.), mounted on Vectashield (Vector Laboratories, Inc., Burlingame, Calif., USA), and observed by fluorescent microscopy (Eclipse E800; Nikon Instruments, Inc., Melville, N.Y., USA). DENV-2-Neutrophil Co-Culture To analyze the effect of DENV-2 on the release of NETs, neutrophils (3 × 105) were seeded on Lab-Tek tissue chambers in 100 μl of RPMI culture medium and incubated for 1 h with DENV-2 at a multiplicity of infection of 10, after which the neutrophils were stimulated with 100 ng/ml of PMA. Neutrophils cultured with DENV-2 only and with PMA only were used as controls. The neutrophils were incubated at 37° in a 5% CO2 atmosphere for 3 h, fixed with 4% paraformaldehyde for 20 min, washed twice with PBS, stained with DAPI (Thermo Fisher Scientific, Inc.), mounted on Vectashield (Vector Laboratories, Inc.), and observed under an Eclipse 800 fluorescent microscope (Nikon Instruments, Inc.). The effect of DENV-2 on the cell viability of Polymorphprep-enriched neutrophils (PMN) was assessed by the exclusion of 7-amino-actinomycin D (7-AAD; BD Biosciences, San Jose, Calif., USA) and flow cytometry. Cells (1 × 106) were incubated with 0.25 μg of 7-ADD at room temperature 10 min before flow cytometry analysis, as indicated by the manufacturer. Quantification of NETs Fluorescent microscopy images of neutrophils under the different culture conditions were analyzed with ImageJ software, as previously described [26]. Briefly, the DAPI signal (cell DNA) area was delineated for each individual cell (200 cells per condition). The mean DNA area was obtained from 3 independent experiments and compared between the different experimental conditions. The DNA area is indicative of the formation of NETs. The results are represented as cell area (arbitrary units).

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nate responses and acute inflammation in the host response to viruses. Likewise, recruitment of neutrophils to the mouse liver follows a viral challenge [8]. However, the role of neutrophils and NETs in viral infections seems to be a double-edged sword. Whereas, for instance, neutrophils recognize HIV-1 by TLR-7 and TLR8, and the engagement of these TLRs triggers the release of NETs that capture HIV-1 and promote HIV-1 elimination through myeloperoxidase and α-defensin [9], hantaviruses stimulate neutrophils by binding to β2integrin receptors such as the complement receptors 3 (CR3) and 4 (CR4), releasing NETs that are thought to contribute to hantavirus-induced immunopathology, leading to kidney and lung damage [10]. In addition, some viruses such as influenza H5N1, West Nile virus, human cytomegalovirus, and Epstein-Barr virus may replicate in neutrophils and, in some instances, neutrophils may even serve as vehicles for virus dissemination to other tissues [11–17]. Dengue is an acute infectious disease caused by any of the four serotypes of dengue virus (DENV-1, -2, -3 or -4), characterized by biphasic fever, headache, body pain, prostration, rash, lymphadenopathy, and leukopenia. Severe cases, known as dengue hemorrhagic fever and dengue shock syndrome, are characterized by increased vascular permeability [18] in which neutrophils may have an important role [19]. In this regard, increased levels of IL-8 have been found in patients with severe DENV infection [19, 20]. IL-8 is a chemokine that attracts neutrophils [21], increases microvascular endothelial cell permeability [22], and stimulates NET formation [23]. Here, we analyzed whether neutrophils release NETs in response to DENV-2 in vitro and found that human peripheral blood-derived neutrophils co-cultured with DENV-2 do not release NETs. On the other hand, when neutrophils were co-cultured with DENV-2 for 1 h and then stimulated with phorbol myristate acetate (PMA), a known inducer of NETs [24], the neutrophils also failed to produce NETs to the same extent as PMA stimulation alone. DENV-2 actually inhibited NET formation by about 80%. As for the possible mechanism, we found that the glucose uptake was significantly lower in DENV2-treated and PMA-stimulated neutrophils compared to PMA-stimulated neutrophils. However, no significant difference in the amount of reactive oxygen species (ROS) was observed, thus suggesting that DENV-2 interferes with NET formation by partially inhibiting glucose uptake, which is part of the metabolic requirement for the release of NETs [25].

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Fig. 1. DENV-2 does not induce the formation of NETs to the same extent as other stimuli. PMN were left untreated or stimulated with 100 ng/ml of PMA as negative and positive controls for NET formation, respectively. In addition, PMN cells were exposed to S. aureus or DENV-2. After the indicated times, cells were fixed with 4% paraformaldehyde, washed with PBS, treated with DAPI for

DNA staining, and then analyzed by fluorescence microscopy (formation of NETs). NETs were already observed at 1 h post-PMA stimulation and at 2 h post-S. aureus stimulation. However, 4 h after DENV-2 stimulation only a few NETs were observed. Results are representative of multiple microscopic fields from 5 independent experiments.

Quantification of Cell Membrane Glut-1 After the indicated treatments, the neutrophils were incubated with PE-labeled rabbit anti-Glut-1 polyclonal antibodies (R&D, Minneapolis, Minn., USA) for 30 min at 4°. After washing, the cells were fixed with 4% paraformaldehyde in PBS. The expression of Glut-1 on the neutrophil cell membrane was assessed by flow cytometry (FACSCalibur; BD Biosciences). Raw data were analyzed with CellQuest software (BD Biosciences). The percentage of Glut1high neutrophils was determined.

diacetate, acetyl ester (CM-H2DCFDA; Thermo Fisher Scientific, Inc.) and flow cytometry. The neutrophils (1 × 106) were suspended in 500 μl of PBS and then labeled with 20 μM CM-H2DCFDA for 20 min at 37° in a 5% CO2 atmosphere. They were then washed with PBS and suspended in RPMI-1640 culture medium and incubated at 37°. At the indicated times, the samples were analyzed by flow cytometry (FACSCalibur; BD Bioscience) with excitation at 488 nm and emission at 525 nm. The raw data were analyzed with CellQuest software (BD Biosciences). In addition, the production of H2O2, specifically, was assessed using the peroxidase-phenol red reagent, as described by Pick and Keisari [27]. Neutrophils, suspended in PBS-G, were deposited in 96well microplates at a density of 5 × 105 cells/well and incubated for 30 min at 37° in a 5% CO2 atmosphere to allow the adhesion of neutrophils to the plastic surface. Then, PBS-G was carefully removed and replaced with 100 μl of peroxidase-phenol red. The neutrophils were: (1) kept unstimulated, (2) stimulated with 100 ng of PMA, (3) stimulated with DENV-2 or (4) preincubated for 30 min with DENV-2 and then stimulated with 100 ng of PMA. Reagents and culture media were included in the assay for the correction of results. Next, the neutrophil preparations were incubated for 2 h at 37° in a 5% CO2 atmosphere. The reaction was stopped by adding 20 μl of 1 N NaOH to each well, and optical density was read at 600 nm in an ELISA plate reader (LabSystems, Melbourne, Vic., Australia).

Glucose Uptake Assays Neutrophils (1 × 106) cultured in serum-free RPMI medium in siliconized glass tubes were supplemented with the fluorescent glucose analog 2-(N-(7-nitrobenzene-2-oxa-1,3-diazol-4-yl) amino)-2-deoxyglucose (2-NBDG; Thermo Fisher Scientific, Inc.) at a final concentration of 250 μM. Then, the neutrophils were either left untreated (medium) or incubated with PMA, DENV-2, or DENV-2 + PMA at 37° for 2 h. After this time, the cells were washed with PBS and fixed with 4% paraformaldehyde in PBS before flow cytometry analysis (FACSCalibur; BD Biosciences). Raw data were analyzed with CellQuest software (BD Biosciences). The mean fluorescence intensity (MFI), indicative of glucose uptake, was normalized for each experiment, taking the MFI value of cells cultured in medium alone as 1.0. The results are represented as the mean fold change in glucose uptake ± SD. ROS Production ROS production, including H2O2, HO, ROO, and ONOO–, was assessed in nonstimulated and PMA-stimulated neutrophils in the presence or absence of DENV-2, using the general ROS probe 5-(and-6)-chloromethyl-2′,7′-dichlorodihydrofluorescein

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Statistical Analyses The percentage of Glut-1high and glucose uptake was analyzed by two-way ANOVA with the normalized data from the MFI. ROS production was analyzed by one-way ANOVA. Analyses were performed using GraphPad Prism Software (La Jolla, Calif., USA). Statistical significance was set at p < 0.05.

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PMA 1 h

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2h

1h

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a

Fig. 2. DENV-2 induces chromatin decondensation in neutrophils. PMN were exposed to DENV-2 for up to 6 h. a At the indi-

cated times, cells were fixed with 4% paraformaldehyde, washed, and treated with DAPI for DNA staining, and then analyzed by fluorescence microscopy. Between 4 and 6 h some cell clumps are observed. This coincides with the release of a few NETs (arrows). However, from 2 h after DENV-2 treatment, most cells present

Results

DENV-2 Does Not Induce the Release of NETs to the Same Extent as Other Stimuli PMA is one of the best pharmacological inducers of NETs [28]. Accordingly, we used PMA (100 ng/ml) as a positive control for the induction of NETs, as previously shown [25]. Neutrophils were stimulated with Staphylococcus aureus, as an additional positive control, and then we proceeded with the analysis of the effect of DENV-2 on NET formation. Figure 1 shows that, as expected, PMA induced the formation of NETs as early as 1–2 h after stimulation, and the same holds true for S. aureus stimulation. On the other hand, DENV-2 did not induce NETs at those times, and up to 4 h of incubation were required to observe only a few NET-producing cells.

DENV-2-Mediated Inhibition of NETs

M1

b

7-AAD

diffuse nuclei, indicative of chromatin decondensation. b Cell viability of DENV-2-treated PMN was assessed by exclusion of 7-AAD staining and flow cytometry. M1 represents the percentage of dead cells. Results are representative of multiple microscopic fields from 5 independent experiments and from two 7-AAD assays.

DENV-2 Induces Chromatin Decondensation in Neutrophils In analyzing NET formation in DENV-2-stimulated neutrophils, we noticed that their nuclei became diffuse by 2 h after the stimuli, a trend that was more evident at 4 and 6 h after the stimuli (fig. 2a). This nucleus morphology is consistent with the previous observation that upon PMA stimulation, in the absence of metabolic substrates (glucose and glutamine), neutrophils undergo chromatin decondensation but are unable to release NETs [25]. The possibility that the lack of more NETs was due to DENV-2-mediated cell lysis was ruled out by 7-AAD staining and flow cytometry, as shown in figure 2b. DENV-2 Inhibits PMA-Induced Formation of NETs When neutrophils were co-cultured with DENV-2 for 1 h, before the addition of 100 ng/ml PMA, the amount of released NETs was lower than that of PMA-activated Intervirology 2015;58:250–259 DOI: 10.1159/000440723

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1.3%

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a p < 0.001 p < 0.001

Fig. 3. DENV-2 partially inhibits PMA-in-

0

DENV-2 and PMA-Induced Expression of Glut-1 It has previously been shown that the formation of NETs is dependent on glucose and the activation-induced increase of the glucose transporter Glut-1 on the neutrophil cell membrane [25]. In order to analyze Intervirology 2015;58:250–259 DOI: 10.1159/000440723

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whether the DENV-2-mediated inhibition of NET formation has some relation with Glut-1, the percentage of Glut-1high neutrophils was assessed by flow cytometry in nonstimulated and PMA-stimulated neutrophils in the presence or absence of DENV-2 at 2 h after PMA stimulation. Figure 4 shows a representative set of flow cytometry raw data. Apparently, DENV-2 treatment before PMA stimulation diminished the percentage of Glut-1high neutrophils compared to PMA stimulation alone. The same pattern was observed in 6 out of 6 independent experiments. However, there was no statistical significant difference between PMA and DENV-2 + PMA. Moreno-Altamirano  et al.  

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b

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neutrophils (fig. 3a).To quantify the actual extent of NET formation, fluorescent microscopy images, as seen in figure 3a, were analyzed with ImageJ software as described [26], and results are shown in figure 3b. Collectively, these results suggest that DENV-2 interferes with the capacity of neutrophils to form NETs in response to PMA stimulation.

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p < 0.001

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duced NET formation. Neutrophils were either left untreated, stimulated with 100 ng/ml PMA, stimulated with DENV-2, or prestimulated with DENV-2 for 1 h and then stimulated with 100 ng/ml PMA. a After 2 h of PMA stimulation, cells were fixed with 4% paraformaldehyde, washed, and treated with DAPI for DNA staining, and then analyzed by fluorescence microscopy for NET formation. b Fluorescence microscopy images were analyzed with ImageJ software to quantify the extent of NET formation.

DENV-2

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DENV-2 Inhibits PMA-Induced Glucose Uptake Glucose uptake is dependent on the amount of glucose transporter on the cell membrane, such as Glut-1 in neutrophils [29]. Therefore, after finding that DENV-2 tends to diminish the percentage of PMA-induced Glut-1high neutrophils compared to PMA stimulation (although the difference was not statistically significant), glucose uptake was evaluated by means of a fluorescent glucose analog (2-NBDG) and flow cytometry. Figure 5a shows a representative dot plot depicting 2-NBDG uptake under the different culture conditions tested, and figure 5b shows the normalized data from 5 independent experiments, expressed as the fold change of 2-NBDG uptake. There was a statistically significant increase in glucose (2-NBDG) uptake when neutrophils were stimulated with PMA. DENV-2-Mediated Inhibition of NETs

26.41%

Glut-1

Fig. 4. DENV-2 and PMA-induced Glut-1high PMN. FSC = For-

p < 0.05 Glucose uptake (fold change)

ward size scatter. The percentage of Glut-1high PMN was assessed by staining neutrophils with an anti-Glut-1 fluorochrome-labeled monoclonal antibody and flow cytometry. Representative dot plot depicting cell size (forward size scatter) versus Glut-1. The Glut1high PMN subpopulation was set from the PMN cultured in medium alone and, using the same gating, Glut-1high PMN cells were calculated in the other PMN culture conditions. The percentage of Glut-1high PMN is depicted in each dot plot. In 6 out of 6 independent experiments DENV-2 treatment diminished the percentage of Glut-1high PMN compared to PMA-stimulated PMN. However, there was no statistically significant difference between these two groups.

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Fig. 5. DENV-2 inhibits PMA-induced glucose uptake by PMN

cells. FSC = Forward size scatter. PMN cells were either left untreated (medium) or cultured with PMA, DENV-2, or DENV-2 + PMA. a Glucose uptake was assessed using the fluorescent glucose analog 2-NBDG, added at the time of PMA stimulation. MFI, indicative of glucose uptake, was quantified by flow cytometry. b Data were normalized, taking the MFI value of cells cultured in medium alone as 1.0. Results are represented as the mean fold change in glucose uptake ± SD at 2 h post-PMA addition (n = 5).

However, this effect was lost when neutrophils were cocultured for 1 h with DENV-2 before PMA addition. In some instances, the glucose uptake was lower in neutrophils exposed to DENV-2 compared to neutrophils cultured in medium alone. Intervirology 2015;58:250–259 DOI: 10.1159/000440723

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Fig. 6. DENV-2 does not interfere with PMA-induced ROS formation in PMN cells. FSC = Forward size scatter; SSC = side size scatter; n.s. = nonsignificant. Isolated neutrophils were left untreated, stimulated with 100 ng/ml PMA, stimulated with DENV-2, or stimulated with DENV-2 and then with 100 ng/ml PMA. ROS (H2O2, HO, ROO, and ONOO–) production was determined by loading cells with CM-H2DCFDA and flow cytometry, and H2O2 by the peroxidase-phenol red reagent and optical density measure-

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c

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ment. a Representative dot plots depicting cell size (forward scatter) versus granularity (side scatter) of isolated PMN cells and gating (R1), and FSC versus ROS (CM-H2DCFDA fluorescence). b Fold change of ROS production, normalizing the flow cytometry data. c H2O2 production (optical density) by peroxidase-phenol red reagent. Results are from 4 and 3 independent experiments, respectively.

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Discussion

Dengue is a major human health concern: about 3 billion people are at risk of infection, since DENV is endemic in more than 100 countries. Dengue shows itself in a wide spectrum of clinical manifestations, ranging from mild fever (dengue fever) to the life-threatening dengue hemorrhagic fever and dengue shock syndrome [31]. Although the mechanisms responsible for one or other clinical outcome are not completely understood, it is clear that plasma/vascular leakage is a hallmark of the most severe forms of the disease [32], which correlates with high serum levels of IL-8 [20], a chemokine that attracts neutrophils and contributes to their degranulation [33]. IL-8 is also a stimulus for the formation of NETs [23]. There is evidence of a direct influence of neutrophils on the adaptive immune response to viral infections [17, 34], and several viruses such as influenza virus H5N1, West Nile virus, human cytomegalovirus, and EpsteinBarr virus may replicate into neutrophils and be transported to other tissues by them [17], whereas several viruses such as HIV-1, influenza, feline leukemia, and rabbit poxvirus have been shown to induce the formation of NETs [35]. Whether neutrophils are infected by DENV remains controversial [36], and whether DENV induces the formation of NETs has not been previously reported. Based on all these findings, the interplay between DENV, neutrophils, and NETs deserves to be analyzed in more detail. Here, we present evidence that DENV-2 does not induce the formation of NETs in vitro to the same extent as other stimuli such as PMA or S. aureus. On the other hand, when neutrophils were co-cultured with DENV-2 and then stimulated with PMA, the formation of NETs DENV-2-Mediated Inhibition of NETs

was inhibited by about 80% compared to PMA stimulation alone. This is, to the best of our knowledge, the first report on DENV-2-mediated inhibition of NETs. Other microorganisms such as HIV-1, Bordetella pertussis, and Cryptococcus neoformans have been shown to inhibit NET formation [9, 37, 38]. C. neoformans and its capsular component GXM (glucuronoxylomannan) inhibit the production of NETs, whereas the acapsular mutant strain CAP67 and the capsular polysaccharide GXM Gal (glucuronoxylomannogalactan) induce NET production [38], suggesting that the mechanism of inhibition is dependent on the engagement of cell membrane receptors. B. pertussis adenylate cyclase toxin suppresses NET formation by generating cAMP and, consequently, inhibiting the oxidative burst [37], and HIV-1 induces the production of IL-10, which may suppress ROS production, thus inhibiting the ROS-dependent NET formation [9]. Recent work has shown that NET release is dependent on glucose uptake and glycolysis, as well as on the increased amount of cell membrane glucose transporter Glut-1 on activated neutrophils [25]. Therefore, we sought to explore whether DENV-2-mediated inhibition of NET formation is related to glucose uptake. The percentage of Glut-1high neutrophils was assessed on activated neutrophils through flow cytometry. The percentage of Glut-1high neutrophils was consistently lower (in 6/6 independent experiments) in DENV-2-exposed and PMA-stimulated neutrophils compared to PMA-stimulated neutrophils in the absence of DENV-2, although the difference was not statistically significant. The uptake of glucose was significantly lower in DENV-2-pretreated and PMA-stimulated neutrophils compared to PMA-stimulated cells, thus suggesting that the cell availability of a metabolic substrate (glucose in this case) required for NET formation to take place is regulated by DENV-2. When ROS production was assessed by two different methods, no difference between DENV-2/ PMA- and PMA-stimulated neutrophils was observed, suggesting that the mechanism by which DENV-2 inhibits the formation of NETs is not related to ROS production. Interestingly, as shown in figure 2a, when neutrophils were exposed to DENV-2, they lost their characteristic multilobulated nuclear morphology, and nuclei became more diffuse, resembling cells at early stages of NET formation or, on the other hand, activated neutrophils cultured in the absence of metabolic substrates [25]. The possibility that DENV-2 induces cell lysis was ruled out by the exclusion of 7-AAD (fig. 2b). It has been proposed that NET formation can be metabolically divided into two phases: chromatin decondensation, which Intervirology 2015;58:250–259 DOI: 10.1159/000440723

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DENV-2 Has No Effect on PMA-Induced ROS Production The production of ROS is required for the formation of NETs [26, 30]. The observation that DENV-2 interferes with the PMA-induced formation of NETs and with the glucose uptake prompted us to ask whether DENV-2 also interferes with ROS production. For this, two different methodological approaches were used. Figure 6a and b shows ROS (H2O2, HO, ROO, and ONOO–) production, as assessed by loading cells with the ROS probe CMH2DCFDA and flow cytometry, and figure 6c shows H2O2 production as assessed with the peroxidase-phenol red reagent [27]. In both cases, it became clear that DENV-2 did not interfere with PMA-induced ROS.

is independent of metabolic substrates, and NET release, which depends on glucose (and glycolysis) and, to a lesser extent, on glutamine [25]. Taken together, the results presented here are consistent with a mechanism in which DENV-2 interferes with the second but not with the first metabolic phase in NET formation (by lowering the uptake of glucose) and suggest a new metabolism-based mechanism for the pathogen-mediated suppression of NET formation.

Acknowledgments The authors thank the Immunology postgraduate instruments core laboratory staff for the use of its facilities, and Sergio Islas-Trujillo for his technical assistance. This work was financed in part by grants from SIP-IPN (20150448) and CONACYT (CB2010-01-158340). O. Rodríguez-Espinosa was the recipient of a CONACYT scholarship. O. Rojas-Espinosa, M.M.B. MorenoAltamirano, and F.J. Sánchez-García are COFAA/EDI/SNI fellows. B. Pliego-Rivera is an SNI fellow.

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Dengue Virus Serotype-2 Interferes with the Formation of Neutrophil Extracellular Traps.

Neutrophils play an important role in the control of pathogens through several mechanisms, including phagocytosis and the formation of neutrophil extr...
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