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. Author manuscript; available in PMC: 2015 Aug 15.
Published in final edited form as: J Immunol. 2014 Jul 11;193(4):1864–1872. doi: 10.4049/jimmunol.1400091

Platelet activation and apoptosis modulate monocyte inflammatory responses in dengue

Eugenio D Hottz 1,2,3,*, Isabel M Medeiros-de-Moraes 1,*, Adriana Vieira-de-Abreu 1,3, Edson F de Assis 1, Rogério Vals-de-Souza 2, Hugo C Castro-Faria-Neto 1, Andrew S Weyrich 3,4, Guy A Zimmerman 1,3, Fernando A Bozza 2,&, Patrícia T Bozza 1,&
PMCID: PMC4137323  NIHMSID: NIHMS605537  PMID: 25015827

Abstract

Background

Dengue is the most prevalent human arbovirus disease in the world. Dengue infection has a large spectrum of clinical manifestations from self-limited febrile illness to severe syndromes accompanied by bleeding and shock. Thrombocytopenia and vascular leak with altered cytokine profiles in plasma are features of severe dengue. Although monocytes have been recognized as important sources of cytokines in dengue, the contributions of platelet-monocyte interactions to inflammatory responses in dengue have not been addressed.

Patients/Methods

Patients with dengue were investigated for platelet-monocyte aggregate formation and markers of monocyte activation. Platelet-induced cytokine responses by monocytes and underlying mechanisms were also investigated in vitro.

Results

We observed increased levels of platelet-monocyte aggregates in blood samples from patients with dengue, especially patients with thrombocytopenia and increased vascular permeability. Moreover, the exposure of monocytes from healthy volunteers to platelets from patients with dengue induced the secretion of the cytokines IL-1β, IL-8, IL-10 and MCP-1, while the exposure to platelets from healthy volunteers only induced the secretion of MCP-1. In addition to the well-established modulation of monocyte cytokine responses by activated platelets through P-selectin binding, we found that interaction of monocytes with apoptotic platelets mediate IL-10 secretion through phosphatidylserine recognition in platelet-monocyte aggregates. Moreover, IL-10 secretion required platelet-monocyte contact but not phagocytosis.

Conclusions

Together, our results demonstrate that activated and apoptotic platelets aggregate with monocytes during dengue infection and signal specific cytokine responses that may contribute to the pathogenesis of dengue.

Keywords: dengue, platelet-monocyte aggregates, cytokines, platelet activation, platelet apoptosis

Introduction

Dengue is the most important arthropod-borne viral disease in the world with over 2.5 billion people living in areas at risk of transmission. Disease is caused by four serotypes of dengue virus (DENV-1 to -4), resulting in over 90 million of apparent infections annually (1-3). Dengue induces a spectrum of clinical manifestations that range from mild self-limited dengue fever to severe dengue, a life-threatening syndrome associated with increased vascular permeability, hypovolemia, hypotension, bleeding and eventually shock (2, 3). Thrombocytopenia is commonly observed in both mild and severe dengue syndromes and correlates with the clinical outcome (3-7). Although thrombocytopenia is a hallmark of dengue infection, the role played by platelets in the pathogenesis of dengue is not completely understood.

We have previously shown that platelets from patients with dengue have characteristics indicating increased activation and apoptosis (8). It is known that activated platelets mediate inflammatory and immune responses by a variety of mechanisms, including release of cytokines and interactions with leukocytes (9-13). A previous study has reported increased platelet-monocyte aggregation in patients with dengue (14). The phagocytosis of apoptotic platelets from DENV-infected patients by macrophages has also been shown (15). Although interaction with activated platelets and recognition of apoptotic bodies have recognized roles in immunomodulation of mononuclear cells (10, 11, 16), the role played by activated and apoptotic platelets in the modulation of monocyte responses during DENV infection has not been addressed.

It is widely accepted that pro-inflammatory cytokines play a major role in the pathogenesis of dengue (4, 17). Nevertheless, the cytokine network and key regulatory pathways are highly complex, and the mechanisms underlying specific cytokine responses by immune cells during dengue infection are not fully elucidated. Here we show that formation of platelet-monocyte aggregates modulates monocyte activation and cytokine release during dengue infection. Specifically, binding of activated and apoptotic platelets from dengue patients induced the secretion of IL-1β, IL-8, IL-10 and MCP-1 in monocytes. Interactions of monocytes with platelets from heterologous healthy volunteers induced the secretion of MCP-1, but not IL-1β, IL-8 and IL-10. In exploring the mechanisms involved, we evaluated the monocyte responses to agonist-stimulated platelets that showed features of activation and apoptosis. We observed that the release of cytokines depended not only on the P-selectin-mediated adhesion (11, 13), but also on the phosphatidylserine-mediated recognition of apoptotic platelets, which induced IL-10 secretion. Our findings provide new insights regarding inflammatory mechanisms in dengue infection and the biology of platelet-monocyte interactions.

Patients and Methods

Human Subjects

Peripheral vein blood samples were obtained from 25 serologically and molecularly confirmed DENV-infected patients from the Instituto de Pesquisa Clínica Evandro Chagas (IPEC) – FIOCRUZ, Rio de Janeiro, Brazil, whose characteristics are presented in Table I. The average day of sample collection after onset of illness was 3.8±1.5 and the average day of defervescence was 4.6±1.3. Peripheral vein blood was also collected from 19 sex and aged-matched healthy subjects. The study protocol was approved by the Institutional Review Board (IPEC #016/2010; and University of Utah) and the experiments were performed in compliance with these protocols. Written informed consent was obtained from all volunteers prior to any study-related procedure.

Table I. Characteristics of healthy volunteers and DENV-infected patients.

Control (19) Dengue (25)
Age, years 29 (26-34) 33 (29-44)
Gender, male 10 (52.6%) 16 (64%)
Platelet count, ×1,000/mm3 241 (215-262) 128 (95-168)*
Leukocyte count, cells/mm3 6,320 (5,285-7,050) 3,750 (2,900-4,560)*
Monocyte, cells/mm3 426 (327.6-515.2) 412 (340.2-538.9)
Hematocrit, % 39.4 (36.4-41.3) 43.1 (40.1-44.0)*
Albumin, g/dL 3.8 (3.4-4.0) 3.6 (3.4-3.7)
TGO/AST, IU/L 19 (15.8-22.2) 40 (32.5-74)*
TGP/ALT, IU/L 28 (20.8-36.5) 62 (45-99)*
Hemorrhagic manifestations1 – 12 (48%)
Intravenous fluid resuscitation – 11 (44%)
Secondary dengue infection – 24 (96%)
Mild Dengue – 13 (52%)
Mild Dengue with warning Signs2 – 12 (48%)
IgM positive 0 (0%) 20 (80%)
IgG positive 14 (74.7%) 24 (96%)
NS1 positive – 7 (28%)
PCR positive3 – 8 (32%)
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Data are expressed as median (interquartile range) or number (%).

1

Gingival, vaginal, and/or gastrointestinal bleeding, petechiae and exanthema.

2

Abdominal pain or tenderness, persistent vomiting, clinical fluid accumulation, mucosal bleed, and/or increased hematocrit concurrent with rapid decrease in platelet count; according to WHO criteria (3).

3

DENV-4 was detected in all PCR-positive patients.

*

p < 0.05 compared to control.

The cohort was comprised of mild dengue patients from which twelve (48%) presented warning signs diagnosed according to World Health Organization (WHO) guidelines (3). Levels of IgM and IgG specific for DENV E protein were measured in plasma from dengue patients using a standard capture ELISA Kit according to the manufacturer's instructions (E-Den01M and E-Den01G, PanBio). DENV NS1 protein was detected in patient plasma using the NS1 detection Kit according to the manufacturer's instructions (BioRad). Primary and secondary infections were distinguished using IgM/IgG antibody ratio, values for IgM/IgG ratio lower than 1.2 were considered secondary infection as previously reported (18-20). 96% of the patients were found to have secondary DV infection.

Platelet and Monocyte Isolation

Peripheral blood samples were drawn into acid-citrate-dextrose and centrifuged at 200 × g for 20 min to obtain platelet-rich plasma (PRP). Platelets were isolated from PRP and CD45+ leukocytes depleted from platelet preparations as previously described (21, 22). The platelet preparation was re-suspended in medium 199 (M199, Lonza Biologics, Switzerland) and its purity (>99% CD41+) confirmed by flow cytometry. Peripheral blood mononuclear cells (PBMC) were isolated from whole blood after PRP was removed (bottom cell layer after the first centrifugation described above) by Ficoll-Paque (GE Healthcare) gradient centrifugation. The monocyte fraction was isolated by CD14+ selection (Human CD14+ selection beads from StemCell, Easy Sep Technology, UK; or from Miltenyi Biotec, AutoMACS Technlogy, UK) according to manufacturer's instructions. Cell viability (>95%) was assessed by Trypan blue exclusion test. The purity of the preparations (>90% CD14+) was confirmed by flow cytometry.

Flow Cytometric Analyses

Platelet-monocyte aggregates were analyzed as previously described (10). Briefly, whole blood was incubated for 10 min with FACS Lysing Solution (BD Bioscience, CA) and then centrifuged at 500 × g for 15 min. The supernatant was discarded, cells were re-suspended in HT buffer (10 mM HEPES, 137 mM NaCl, 2.8 mM KCl, 1 mM MgCl2.6H2O, 12 mM NaHCO3, 0.4 mM Na2HPO4, 5.5 mM glucose, 0.35% BSA; pH 7.4) and incubated (20 min, RT) in the presence of PE-conjugated anti-CD41 (BD Pharmingen, CA) and FITC-conjugated anti-CD14 (BD Pharmingen, CA). After incubation, 250 μL of FACS Lysing solution was added to fix the samples. To assess platelet activation, freshly isolated platelets were incubated (30 min, RT) with FITC-conjugated anti-CD41 (0.5 μg/mL) and PE-conjugated anti-CD62P (0.25 μg/mL) (BD Pharmingen, CA). Isotype-matched antibodies were used to control nonspecific binding of antibodies. Platelets and monocytes were distinguished by characteristic forward and side scattering and specific binding to CD41 or CD14, respectively.5,000 to 10,000 gated events were analyzed using a FACScalibur flow cytometer (BD Bioscience, CA). Cell surface phosphatidylserine exposure was determined with FITC-conjugated Annexin V (Beckman Coulter, France). Mitochondrial membrane potential (ΔΨm) was measured using the probe tetramethylrhodamine methyl ester (TMRM; Invitrogen) (100 nM, 10 min).

Platelet-Monocyte in vitro interactions

To examine interactions of platelets and monocytes from patients and healthy volunteers, purified heterologous platelets and monocytes were incubated with one another for 12 h at 37°C in 5% CO2 atmosphere. Each experimental point contained 105 monocytes and 107 platelets in a volume of 100 μL of M199 containing 10μg/mL of polymyxin B (Sigma-Aldrich). Platelets and monocytes alone were also examined under the same conditions. Cells were recovered by centrifugation at 500 × g for 10 min, fixed with 4% paraformaldehyde (10 min), and platelet-monocyte aggregates evaluated by flow cytometry as described above. The supernatants from platelets, monocytes and platelet-monocyte aggregates were collected and stored at -20°C until analysis.

For the interactions of agonist-stimulated platelets with monocytes, autologous platelets and monocytes were incubated with one another for 8 h at 37°C in 5% CO2 atmosphere. Each experimental point contained 5×105 monocytes and 5×107 platelets in a volume of 200 μL of M199 containing 10μg/mL of polymyxin B. Platelets were stimulated with thrombin (Sigma-Aldrich, T1063) (0.5 U/mL) or thrombin plus convulxin (Santa Cruz, sc-202554) (250 ng/mL) for 5 min. This platelets were then diluted 1:5 and incubated with monocytes (final concentration of thrombin and convulxin in monocytes were 0.1 U/mL and 50 ng/mL, respectively) in the presence or absence of anti-P-selectin (R&D systems, BBA30) (10μg/mL), anti-phosphatidylserine (abcam, ab18005) (50μg/mL) or isotype matched antibody.

Platelet phagocytosis assay

Platelet phagocytosis was assayed as previously described (23). Briefly, platelets were labeled with the CellTracker Far Red DDAO-SE (Molecular Probes) (5μM) for 1 hour at 37°C, washed three times by resuspending in warm PSG containing 100 nM PGE1 (Cayman Chemicals; Michigan, USA) and centrifuging at 500 × g for 20 min, and resuspended in M199. Labeled platelets were then stimulated with thrombin and/or thrombin together with convulxin as above described, and incubated with monocytes for 1 h at 37°C to allow phagocytosis to proceed. Cells were washed in HBSS, quenched with 0.1% trypan blue in HBSS for 20 minutes, washed once, and analyzed by Flow cytometry. Monocytes incubated with unlabeled platelets and monocytes incubated with labeled, stimulated platelets and kept unquenched were used to set up the flow cytometer. Monocytes treated with the cytoskeleton assembly inhibitors Cytochalasin D (10 μg/mL) and Cytochalasin B (10 μg/mL) were used as negative controls for platelet phagocytosis.

Monocyte adhesion to immobilized P-selectin and phosphatidylserine

Monocytes adhesion assays were performed as previously described (11). Briefly, 300μL of phosphatadylserine (100 μg/ml) dissolved in ice-cold ethanol were added to the wells of flat-bottomed 16-mm plates (Nunclon, Denmark) and incubated for 18 hours at 4°C to evaporate the ethanol. Control wells that were not coated with phohsphatidylserine were treated with ethanol alone. The wells were then incubated overnight at 4°C with HBSS containing HSA or P-selectin (10mg/ml), and blocked with HSA (10 mg/mL) for 4 h at 25°C. The plates were washed twice with HBSS-0.05% Tween-20 and three times with HBSS. 106 monocytes resuspended in 300 μL of M199 medium containing 10 mg/mL of polymyxin B were added to the coated surfaces and maintained at 37°C for 8h. Adherent cells were fixed, stained with Giemsa and counted by light microscopy.

Cytokine measurement

The content of the cytokines FGF-β, G-CSF, GM-CSF, IFN-γ, IL-1β, IL-1Ra, IL-2, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-12p70, IL-13, IL-15, IL-17, IP-10, MCP-1, MIP-1α, MIP-1β, PDGF, RANTES, TNF-α and VEGF in the supernatants from platelet-monocyte interactions was measured using a Multiplex cytokine immunoassay (Bio-Plex Human Cytokine Assay; Bio-Rad, CA). Levels of IL-8 and IL-10 were also determined using standard capture ELISA Kit (R&D systems, MN).

Statistical analysis

Statistical analyses were performed using GraphPad Prism, version 5.0 (GraphPad Software, San Diego, CA, USA). The numerical demographic and clinical variables were expressed as the median and the interquartile range (IQ 25-75%) or number and percent (%). All of the numerical variables were tested for a normality distribution using the Kolmogorov-Smirnov test. We compared the continuous variables using the t-test (parametric distribution) or the Mann-Whitney U test (non-parametric distribution). Correlations were assessed using the Pearson's test.

Results

Increased platelet-monocyte aggregates in patients with dengue

We have previously shown that platelets in the blood of patients with dengue are activated (8). Consistent with this, platelets in samples from dengue patients in the present study also had increased P-selectin surface expression (data not shown). P-selectin is the primary adhesion molecule on activated platelets that binds leukocytes (11, 13, 24). To investigate whether activated platelets can interact with monocytes during active dengue infection, we analyzed platelet-monocyte aggregates in peripheral whole blood samples by flow cytometry. As shown in Figure 1A, dengue patients had increased platelet-monocyte aggregates when compared to healthy volunteers (26.1±14.1% vs 8.1±1.7%, p<0.001). Moreover, platelet P-selectin surface expression positively correlated with the levels of circulating platelet-monocyte aggregates in samples from patients with dengue and healthy volunteers (r=0.69, p<0.01) (Figure 1B).

Figure 1. Increased platelet-monocyte aggregates in dengue illness.

Figure 1

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(A) The percentage (%) of platelet-monocyte aggregates identified as CD14+CD41+ monocytes was assessed in healthy subjects (control) and patients with dengue (dengue). The boxes indicate the median and interquartile ranges and the whiskers indicate the 5-95 percentiles. The asterisk (*) signifies p<0.01 compared to control. The insets show representative density plots for CD41-positive monocytes from one healthy volunteer and one dengue-patient. The values in each region of the quadrants indicate the cell frequencies of the quadrant. (B) The % of platelets with CD62-P surface expression was plotted against the % of CD14+CD41+ monocytes in the same patient or healthy volunteer. Linear regression was traced according to the distribution of points.

Platelet-monocytes aggregates are associated with thrombocytopenia and increased vascular permeability during dengue disease

Using platelet counts determined on the day of sample collection, patients were classified as thrombocytopenic (<150,000/mm3) or non-thrombocytopenic. Based on this grouping, 45% of the patients were thrombocytopenic while 55% were not. Platelet-monocyte aggregates were higher in thrombocytopenic compared to non-thrombocytopenic dengue patients (34.2±18.4 vs 20.3±6.8, p=0.0151) (Figure 2A). The breakdown was similar in patients who were positive or negative for signs of increased vascular permeability. Increased vascular permeability was evidenced by one or more of the following signs: increase in hematocrit greater than 20%, hypoalbuminemia, postural hypotension, ascites and oliguria. According to the presence or absence of these signs 48% of patients were classified as positive and the remaining 52% negative. The percentage of platelet-monocyte aggregates was significantly higher in patients who were positive for signs of increased vascular permeability when compared to patients who did not have evidence of vascular leak (32.7±18.5% vs 20.6±5.3%, p=0.021) (Figure 2B). Moreover, we found that platelet-monocyte aggregates in DENV-infected patients inversely correlated with platelet counts and plasma albumin levels (Figure 2C-D).

Figure 2. Platelet-monocyte aggregates correlate with thrombocytopenia and increased vascular permeability in dengue.

Figure 2

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The percentage (%) of CD14+CD41+ monocytes was assessed in health volunteers (control) and dengue patients (dengue) that were positive (+) or negative (−) for (A) thrombocytopenia or (B) signs of increased vascular permeability. The boxes indicate the median and interquartile ranges and the whiskers indicate the 5-95 percentiles. The asterisk (*) signify p<0.01 compared to control. The pound sign (#) indicates p<0.05 between positive and negative. (C-D) The % of CD14+CD41+ monocytes was plotted against (C) the platelet counts obtained on the same day that platelet-monocyte aggregates were analyzed and (D) the lowest plasma albumin level for each patient. Linear regressions were traced according to the distribution of points

Platelets from dengue-infected patients aggregate with control monocytes and induce cytokine release in vitro

Next we investigated the ability of platelets isolated from patients with dengue to aggregate with monocytes from healthy volunteers and modulate monocyte responses. Increased platelet-monocyte aggregate formation was observed when monocytes from healthy volunteers were exposed to platelets from DENV-infected patients in comparison to platelets from heterologous healthy volunteers (62.5±9.1 vs 30.7±11.6, p=0.006). Incubation of platelets from healthy volunteers with monocytes from dengue patients did not promote any increment in platelet-monocyte aggregates when compared to control platelets plus control monocytes (41.8±19.5, p=0.2234) (Figure 3).

Figure 3. Platelets from dengue-infected patients aggregate with control monocytes in vitro.

Figure 3

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Platelets and monocytes from healthy volunteers (control, C) or patients with dengue (D) were incubated with one another as described. C+C, platelets plus monocytes from heterologous control participants; C+D, control platelets plus monocytes from dengue patients; or D+C, platelets from dengue patients plus control monocytes. The percentage (%) of CD14+CD41+ monocytes is shown. The bars represent mean±SEM of 7 independent platelet plus monocyte combinations. The asterisk (*) signifies p<0.05 compared to C+C. The insets show representative dot plots for CD41-expressing monocytes.

We have previously shown that signaling by activated adherent platelets enhances cytokine and chemokine production by monocytes, including TNF-α, IL-1β, IL-8 and MCP-1 (10, 11, 25). In this study, the levels of IL-1β and IL-8 were significantly (p<0.05) elevated in the supernatant of monocytes exposed to platelets from dengue-infected patients when compared to control platelets (Figure 4A-B). The levels of MCP-1 were elevated in all platelet-monocyte interactions compared to monocytes alone, regardless of patient or control source of the cells (Figure 5C). Also, increased RANTES secretion was observed in platelets from healthy volunteers when compared to dengue-infected patients (Figure 4D).

Figure 4. Platelets from dengue-infected patients modulate the monocyte cytokine profile.

Figure 4

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Platelets and monocytes from healthy volunteers (control, C) or patients with dengue (dengue, D) were incubated alone or with one another as described. C+C, platelets plus monocytes from heterologous control participants; C+D, control platelets plus monocytes from patients with dengue; or D+C, platelets from dengue patients plus control monocytes. The concentrations of (A) IL-1β, (B) IL-8, (C) MCP-1, (D) RANTES, (E) TNF-α, and (F) IL-10 in the supernatants of cells incubated in each condition are shown. The bars represent mean±SEM of 7 independent platelet plus monocyte combinations. The asterisk (*) signifies p<0.05 compared to C+C. The symbol (&) signifies p<0.05 compared to monocytes from the same origin (control or dengue). The pound sign (#) indicates p<0.05 between specified groups.

Figure 5. Apoptotic platelet recognition mediate IL-10 secretion by platelet-monocyte aggregates.

Figure 5

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(A) Representative density plots showing P-selectin surface expression, Δ(X00471)m and phosphatydilserine (PhSer) exposure in platelets activated with thrombin (Thr) and/or convulxin (Cvx). (B-F) Monocytes were exposed to resting platelets or platelets activated with Thr or Thr+Cvx in the presence or absence of neutralizing antibodies against CD62-P or PhSer. The concentrations of IL-8 (B, C and E) and IL-10 (B, D and F) in the supernatants of cells incubated in each condition are shown. The bars represent mean±SEM of 4-8 independent experiments. The asterisk (*) signifies p<0.05 compared to unstimulated platelets. The pound sign (#) indicates p<0.05 compared to IgG.

The secretion of TNF-α was not different between monocytes exposed to platelets from control or dengue subjects or in platelet-monocyte interactions compared to monocytes alone (Figure 4E). Interestingly, we observed that monocytes exposed to platelets from dengue-infected subjects secreted increased levels of IL-10 (Figure 4F), a cytokine not previously demonstrated to be directly modulated by platelet-monocyte binding. Other cytokines measured in the multiplex assay were either below detection limit or not different among platelets, monocytes or platelets-monocyte interactions from patients and controls (data not shown).

P-selectin and phosphatidylserine are required for IL-10 secretion in platelet-monocyte aggregates

We have previously shown increased platelet apoptosis in addition to platelet activation in patients with dengue (8). Platelets from patients in the present study similarly showed increased phosphatidylserine exposure (26.9±7.9% vs 4.9±3.2%, for dengue patients and healthy volunteers, respectively). We hypothesized that monocytes secrete IL-10 (Figure 4F) in response to the recognition of apoptotic platelets in platelet-monocyte aggregates. Thus, we evaluated monocyte responses after exposure to activated platelets or to activated and apoptotic platelets. Platelet activation and apoptosis were induced by specific agonist stimulation as previously described (26). Platelets stimulated with thrombin or convulxin alone became activated but not apoptotic; in contrast, platelets stimulated with thrombin plus convulxin became activated and apoptotic as demonstrated by P-selectin surface expression, phosphatidylserine exposure and loss of ΔΨm (Figure 5A). As shown in Figure 5B, monocytes incubated with thrombin-activated platelets secreted IL-8 but not IL-10. The exposure of monocytes to platelets stimulated with thrombin plus convulxin, on the other hand, induced both IL-8 and IL-10. Importantly, the agonists alone did not induce significant cytokine secretion in monocytes.

To better understand the mechanisms by which monocytes secrete cytokines in response to apoptotic and/or activated platelets, monocytes were exposed to platelets in the presence of anti-Pselectin or anti-phosphatidylserine antibodies. As previously reported (11), blocking of P-selectin damped the secretion of IL-8 in monocytes interacted with activated platelets, independently if stimulated with thrombin or thrombin plus convulxin (Figure 5C). Interestingly, the secretion of IL-10 in monocytes exposed to platelets stimulated with thrombin plus convulxin was also damped by P-selectin blocking (Figure 5D). The secretion of IL-8 by platelet-monocyte aggregates was not affected by anti-phosphatidylserine antibodies (Figure 5E). Nevertheless, blocking of phosphatidylserine on apoptotic platelets significantly reduced the secretion of IL-10 (Figure 5F). These data indicate that IL-10 secretion by platelet-monocyte aggregatation depends on both P-selectin-mediated binding and phosphatidylserine recognition on activated and apoptotic platelets.

Phosphatidylserine recognition is sufficient to induce IL-10 secretion by monocytes

Beyond its immunomodulatory activities, phosphatidylserine recognition is the main signal for apoptotic cell phagocytosis. During platelet-monocyte aggregation, the levels of platelet phagocytosis were higher in monocytes interacting with thrombin plus convulxin-stimulated platelets compared with unstimulated platelets (Figure 6A-B). Treatment of platelets with anti-Pselectin or anti-phosphatidylserine antibodies significantly reduced the phagocytosis of apoptotic platelets (Figure 6A-B). To better understand the role played by platelet phagocytosis in the regulation of IL-10 secretion, monocytes were pretreated (30 min) with the cytoskeleton assembly inhibitors Cytochalasin D (10 μg/mL) and Cytochalasin B (10 μg/mL) which significantly impaired uptake of apoptotic platelets (Figure 6A-B). Interestingly, platelet phagocytosis was not required to the secretion of IL-8 or IL-10 (Figure 6C-D). We next investigated whether a synergistic signaling of P-selectin and phosphatidylserine is required to induce IL-10 synthesis. We observed increased adhesion of monocytes plated on P-selectin and/or phosphatidylserine compared to HAS (Figure 6E-F). Monocytes plated on P-selectin and/or phosphatidylserine also secreted increased levels of IL-8. The secretion of IL-10, however, was preferentially found in monocytes adherent to phosphatidylserine or to P-selectin plus phosphatidylserine, compared with HAS coated plates (Figure 6G). These results indicate that phosphatidylserine recognition is sufficient to induce IL-10 secretion by monocytes, indicating that phagocytosis of apoptotic platelets, or synergistic signaling by P-selectin plus phosphatidylserine, is not required.

Figure 6. Phosphatidylserine recognition mediates platelet phagocytosis and IL-10 secretion.

Figure 6

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(A-B) Representative density plots (A) and bar graphs (B) showing the percent of platelet phagocytosis by monocytes exposed to resting platelets, or platelets stimulated with thrombin or thrombin plus convulxin in the presence or absence of anti-CD62P, anti-phosphatidylserine (PhSer) or cytochalasin D plus cytochalasin B (CytoD+B). (C-D) The concentrations of IL-8 (C) and IL-10 (D) in the supernatants of cells treated with CytoD+B are shown. (E-F) Monocyte adhesion on plates coated with human serum albumin (HSA), P-selectin (CD62P), PhSer, or P-selectin + PhSer. (G) The concentrations of IL-8 and IL-10 in the supernatants of monocytes adherent to each substrate are shown. Panels A and E show representative images and density plots from 4 independent experiments. The values in each region of the quadrants indicate the cell frequencies of the quadrant. The bars represent mean±SEM of 4 independent experiments. The asterisk (*) signifies p<0.05 compared to unstimulated platelets or HAS-coated plates. The pound sign (#) indicates p<0.05 compared to IgG or vehicle (DMSO).

Discussion

Thrombocytopenia and increased vascular permeability are hallmarks of dengue illness. Although high concentration of pro and anti-inflammatory cytokines have been extensively reported in dengue patients (4, 27-29), the sources and determinants for cytokines secretion are not fully elucidated. Our results demonstrate a role for platelet-monocyte interactions in the activation of monocytes during dengue infection. We observed increased levels of platelet-monocyte aggregates in patients with dengue, especially in samples from patients who presented thrombocytopenia and signs of increased vascular permeability. We found evidence that platelet binding modulates cytokine responses by monocytes in dengue. Interaction with platelets from patients with dengue enabled monocytes from healthy volunteers to synthesize and secrete IL-1β, IL-8 and IL-10. Experiments with in vitro stimulated platelets showed that the secretion of cytokines is regulated by P-selectin-mediated adhesion and, in addition, recognition of apoptotic platelets through phosphatidylserine (Summarized in Figure 7). Induction of immunomodulatory gene expression in platelet-monocyte aggregates by phosphatidylserine signaling has not been reported previously.

Figure 7. Schematic representation for platelet-induced cytokine secretion by monocytes.

Figure 7

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(A) Interaction of activated and apoptotic platelets with monocytes during DENV infection promotes secretion of IL-1β, IL-8, IL-10 and MCP-1. (B) Surface binding of P-selectin to PSGL1 and recognition of phosphatidylserine (PhSer) by PhSer receptors, in parallel to the secretion of chemokines from platelet α-granules, are main mechanisms for immunomudalation of monocytes by activated and apoptotic platelets after agonist stimulation or at diseases conditions where platelet activation and apoptosis take place (i.e. dengue).

Platelet adhesion to leukocytes is mediated by platelet P-selectin surface expression (11, 13, 24), which is increased in platelets from patients with dengue (8). Onlamoon and coworkers (30) found that DENV elicits platelet-monocyte and platelet-neutrophil aggregates in a primate model for severe dengue. Platelet-monocyte aggregates have also been observed in mild dengue in humans (14). In these interactions, the binding of P-selectin on activated platelets to P-selectin glycoprotein ligand-1 (PSGL-1) on monocytes not only tethers the cells together but also triggers functional responses in the monocytes (24), among them cytokine synthesis and secretion (10, 11, 25). Of importance, the cytokines IL-1β, IL-8 and IL-10, which were released by monocytes in response to interaction with platelets from dengue patients, are frequently increased in plasma from patients with severe dengue (4, 27-29).

It is known that signals delivered to monocytes by binding of platelet P-selectin to PSGL-1 are integrated and amplified by factors secreted from platelets (11, 31), including the chemokine RANTES (11). In this study, platelets from dengue-infected patients secreted lower levels of RANTES in vitro than platelets from healthy volunteers. This may be explained by extensive release of platelet granule contents in vivo before platelet isolation, since platelets from patients with dengue were shown to be activated. Furthermore, we have previously shown that platelets release RANTES in response to DENV exposure (21). As MCP-1 synthesis by monocytes in response to platelet adhesion depends on concomitant RANTES signaling (11), the exhaustion of RANTES in platelets from dengue infected patients may explain why these platelets failed to induce higher MCP-1 secretion compared to control platelets, even though they express more P-selectin. Platelet-monocyte aggregate formation probably contributes to MCP-1 release in vivo, since both platelet-monocyte aggregates and RANTES are increased in patients with dengue (29, 32).

In previous studies from our group and others (4, 33) (34), the levels of MCP-1, IL-1β, IL-8 and IL-10 were associated with severe thrombocytopenia in dengue. These associations are in agreement with the formation of platelet-monocyte aggregates as a mechanism for both thrombocytopenia and cytokine release, as suggested by the correlation of platelet-monocyte aggregates with platelet counts in the present work. Platelet-monocyte aggregates were also associated with increased vascular permeability in dengue patients. The endothelial effects of the cytokines released in response to platelet-monocyte binding potentially explain this association. The cytokines IL-1β, IL-8 and MCP-1 are reported to be mediators in DENV-triggered endothelial leak based on in vitro assays (21, 35, 36). Furthermore, IL-1β is linked to enhanced vascular permeability, hypotension and hemoconcentration in dengue-infected patients (4, 21). We have previously shown that activated platelets are important sources of IL-1β during dengue infection, and that platelet-released IL-1β contributes to increased endothelial permeability in dengue (21). We now show that activated platelets also contribute to IL-1β levels in dengue by inducing IL-1β synthesis in monocytes. We cannot exclude, however, that IL-1β secreted by platelets from patients with dengue may have contributed to the higher levels of IL-1β in platelet-monocyte interactions (Figure 4A). Relevant to the importance of platelets and platelet-monocyte aggregates as sources of IL-1β in systemic infections, depletion of platelets completely reduced the circulating levels of IL-1β in mice with experimental cerebral malaria (37).

Beyond pro-inflammatory cytokines, exposure to platelets from dengue infected patients induced the secretion of IL-10 in monocytes. IL-10 is an anti-inflammatory and regulatory cytokine with many immunomodulatory properties. This cytokine has been shown to inhibit DENV-specific T cell responses as shown by ex vivo and in vitro models (38). Accordingly, patients with severe dengue present increased levels of IL-10 than patients with mild dengue (39)(40), and higher levels of IL-10 are observed in non-survivals than in survivals among severe dengue patients (41).

In addition to platelet activation, we have previously shown that platelets from dengue patients have characteristics indicative of apoptosis, among them the exposure of phosphatidylserine (8). Similarly, platelets from dengue patients in the present study had increased phosphatidylserine exposure. Phosphatadylserine exposure is the main “eat-me” signaling for apoptotic cell clearance. Recognition of phosphatidylserine by mononuclear phagocytes not only promotes apoptotic cell uptake but also immunoregulatory responses including IL-10 synthesis and secretion (16, 42). Alonzo and colleagues (15) have previously shown that cultured macrophages phagocytized platelets from dengue-infected patients depending on phosphatidylserine recognition. In our model for agonist-induced platelet apoptosis, the recognition of phosphatidylserine not only induced platelet phagocytosis but also IL-10 secretion. IL-10 secretion was also induced in monocytes exposed to platelets from patients with dengue. Gudbrandsdottir and colleagues (43) have recently reported higher IL-10 synthesis in platelet-attached monocytes after stimulation of PBMC with LPS in the presence of platelets. In agreement, platelet phagocytosis and IL-10 secretion were also influenced by P-selectin-mediated binding in our platelet-monocyte interactions. Although blocking P-selectin reduced IL-10 secretion in platelet-monocyte aggregates, our results with isolated adherent monocytes indicate that the recognition of phosphaidylserine is sufficient to induce IL-10 secretion. P-selectin binding probably contributes to apoptotic platelet phagocytosis and IL-10 secretion by facilitating phosphatidylserine recognition while tethering the cells together. These results are in agreement with others who showed that the apoptotic platelet clearance by neutrophils is a biphasic event depending firstly on P-selectin-mediated adhesion followed by phosphatidylserine-mediated internalization (44). Thus, P-selectin and phsphatidylserine mediated cell interactions may represent new targets for dengue treatment research, and antibodies against P-selectin and phosphatidylserine were recently shown to be safe for human use in phase 1 clinical trials (45)(46)(47). While our findings suggest that monocytes secrete IL-10 through recognition of apoptotic platelets in dengue, we cannot exclude that this also occurs in response to other apoptotic cell types. In a previous study, levels of IL-10 were associated with the presence of apoptotic lymphocytes in dengue-infected patients (39).

In summary, we report for the first time the contributions of platelets to inflammatory and immunemodulatory responses of monocytes during dengue infection. We provide evidence for platelet-monocyte aggregate formation during dengue infection and platelet-dependent monocyte activation with IL-1β, IL-8, IL-10 and MCP-1 synthesis and secretion. We also provide new insights regarding the biology of platelet-monocyte interactions, with the recognition of phosphatidylserine on apoptotic platelets as a key immuneregulatory event. Each of these events and cellular interactions potentially contribute to the pathogenesis of dengue.

Acknowledgments

We thank Robert Campbell for technical assistance; and the PDTIS (Programa de Desenvolvimento Tecnológico em Insumos para Saúde) platform for multiplex analysis.

Funding: This work was supported by grants from Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Fundação de Amparo a Pesquisa do Estado do Rio de Janeiro (FAPERJ), Programa Estratégico de Apoio à Pesquisa em Saúde (PAPES)/FIOCRUZ, and Programa de Apoio a Núcleos de Excelência (PRONEX) Dengue; and from the National Institutes of Health (NIH) (HL066277, HL091754, and R37HL044525 awarded to A.S.W. and G.A.Z). Dr. Guy Zimmerman is the recipient of a Ciência Sem Fronteiras special visiting professorship from CNPq.

Abbreviations used in this article

DENV

dengue virus

IL

interleukin

MCP

monocyte chemoattractant protein

TNF

tumor necrosis factor

RANTES

regulated upon activation normal T cell expressed and secreted

WHO

World Health Organization

PBMC

peripheral blood mononuclear cells

ELISA

enzyme-linked immunosorbent assay

HAS

human serum albumin

PSGL

P-selectin glycoprotein ligand

LPS

lipopolysaccharide

Footnotes

Author contributions: E.D.H. and I.M.M.M.– Performed the majority of experiments, data analyses, and manuscript drafting and preparation; E.F.A. – performed part of experiments and analyzed data; R.V-d-S.- Patient enrollment, clinical classification and experimental analyses; A.V.A. – Experimental design and manuscript editing/review; H.C.C-F-N. - Experimental design and manuscript editing/review; A.S.W.- Experimental design and manuscript editing/review; G.A.Z.- Experimental design and manuscript editing/review; P.T.B. and F.A.B.- Directed all aspects of the study, data analyses, and manuscript preparation and review.

Conflict-of-interest disclosure: The authors declare no competing financial interests.

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