Platelets mediate increased endothelium permeability in dengue through NLRP3-inflammasome activation

Abstract

Dengue is the most frequent hemorrhagic viral disease and re-emergent infection in the world. Although thrombocytopenia is characteristically observed in mild and severe forms of dengue, the role of platelet activation in dengue pathogenesis has not been fully elucidated. We hypothesize that platelets have major roles in inflammatory amplification and increased vascular permeability during severe forms of dengue. Here we investigate interleukin (IL)-1β synthesis, processing, and secretion in platelets during dengue virus (DV) infection and potential contribution of these events to endothelial permeability during infection. We observed increased expression of IL-1β in platelets and platelet-derived microparticles from patients with dengue or after platelet exposure to DV in vitro. We demonstrated that DV infection leads to assembly of nucleotide-binding domain leucine rich repeat containing protein (NLRP3) inflammasomes, activation of caspase-1, and caspase-1-dependent IL-1β secretion. Our findings also indicate that platelet-derived IL-1β is chiefly released in microparticles through mechanisms dependent on mitochondrial reactive oxygen species-triggered NLRP3 inflammasomes. Inflammasome activation and platelet shedding of IL-1β-rich microparticles correlated with signs of increased vascular permeability. Moreover, microparticles from DV-stimulated platelets induced enhanced permeability in vitro in an IL-1-dependent manner. Our findings provide new evidence that platelets contribute to increased vascular permeability in DV infection by inflammasome-dependent release of IL-1β.

Figure 1

IL-1β synthesis in DV-infected platelets and platelets from healthy volunteers. (A) The percentage of IL-1β–expressing platelets was assessed in platelets that were isolated from healthy subjects (control) or patients with dengue (dengue). The boxes indicate the median and interquartile ranges and the whiskers indicate 5 to 95 percentile. (B-C) Washed platelets from healthy volunteers were exposed to mock, infective DV-2 (DV) or heat-inactivated DV-2 (heat DV) as described in Patients and methods. Panel B shows the percentage of IL-1β–expressing platelets, and panel C shows the concentration of IL-1β in supernatants of platelets incubated in each condition. The bars represent mean ± standard error of the mean (SEM) of 8 experiments performed using samples from independent healthy donors. *P < .05 and **P < .01 compared with healthy volunteers or mock exposed platelets. (Insets) Representative density plots of (A) IL-1β–expressing platelets from 1 healthy volunteer and 1 dengue patient or (B) platelets from 1 healthy volunteer that were exposed to mock, DV, or heat DV. The values in each region of the quadrants indicate the cell frequencies in the respective quadrant.

Figure 2

Dengue induces shedding of IL-1β–rich microparticles in platelets. (A) The proportion of MPs per 100 platelets was assessed in samples from healthy subjects (control) or dengue-infected patients (dengue). (B) The percentage of IL-1β–positive MPs in samples obtained from control participants or dengue patients. The boxes indicate the median and interquartile ranges and the whiskers indicate 5 to 95 percentile. (C-D) Washed platelets from healthy volunteers were exposed to mock, infective DV-2 (DV), or heat-inactivated DV-2 (heat DV) as described in Patients and methods. (C) The levels of platelet-derived MPs and (D) the percentage of IL-1β–containing MPs for each condition are shown. The bars represent mean ± SEM of 8 experiments performed using samples from independent healthy donors. *P < .05 and **P < .01 compared with healthy volunteers or mock exposed platelets. (Insets) Representative density plots showing (B) IL-1β–expressing MPs from 1 healthy volunteer and 1 dengue patient or (D) platelets from 1 healthy volunteer that were exposed to mock, DV, or heat DV. The values in each region of the quadrants indicate the cell frequencies in the respective quadrant.

Figure 3

Activation of the NLRP3 inflammasome in platelets from DV-infected patients. (A-B) Representative density plots showing the expression of NLRP3 in (A) platelets and (B) MPs from healthy subjects (control) or patients with dengue (dengue). The values in each region of the quadrants indicate the cell frequencies in the respective quadrant. (C) Platelets obtained from healthy subjects and patients with dengue were stained with anti-NLRP3 (green) and anti-ASC (red) and examined by confocal microscopy using an Olympus FV10i-O with a 240× lens. The images were processed using FLUOVIEW FV1000 MPE software version 5.0 and further analyzed using Adobe Photoshop CS Version 8.0. Bars represent 10 µm. (D) Caspase-1 activation in platelets isolated from control participants and dengue patients was assessed by the MFI of the fluorescent probe FLICA. The boxes indicate the median and interquartile ranges and the whiskers indicate 5 to 95 percentile. **P < .01 compared with healthy volunteers. (E) Western blot analysis of pro (p31) and cleaved (p17) IL-1β and of β-actin in platelets that were isolated from 3 healthy volunteers and 3 dengue patients.

Figure 4

Platelet shedding of IL-1β–rich MPs depends on caspase-1 activation. (A) Platelets from healthy subjects were exposed to mock, infective DV-2 (DV), or heat-inactivated DV-2 (heat DV), and caspase-1 activation was evaluated through the MFI of the fluorescent probe FLICA. (B-D) Platelets were exposed to mock, DV, or heat DV in the presence or absence of the caspase-1 inhibitor YVAD. B depicts the percentage of IL-1β–expressing platelets, and panel C shows the concentration of IL-1β in the supernatant of platelets incubated in each condition. Panel D shows the percentage of IL-1β–containing MPs for each condition. The bars represent the mean ± SEM of 6 to 8 independent experiments using samples from different healthy donors. *P < .05 and **P < .01 compared with mock exposed platelets that received the same treatment (dimethylsulfoxide [DMSO] or YVAD). ^#P < .05 between DV-2–treated platelets that were incubated with YVAD or DMSO.

Figure 5

DV-elicited inflammasome activation depends on mitochondrial-derived ROS and RIP kinases. Platelets from healthy subjects were exposed to mock, infective DV-2 (DV), or heat-inactivated DV-2 (heat DV) in the presence or absence of the mitochondrial targeted antioxidant mitoTEMPO or the RIP1 inhibitor necrostatin-1 (Nec-1). Panels A and B show the fold changes for (A) MitoSox Red fluorescence and (B) caspase-1 activation related to mock values. (C-D) The levels of (C) IL-1β and (D) RANTES were determined in the supernatants of platelets incubated in each condition. The bars represent the mean ± SEM of 6 independent experiments using samples from different healthy donors. *P < .05 and **P < .01 compared with mock exposed platelets that were treated with the same drug. ^#P < .05 between DV-2–exposed platelets that were incubated with mitoTEMPO or Nec-1 and platelets that were incubated with DMSO. ND, not detected; ns, nonsignificant.

Figure 6

Vascular permeability is associated to platelet shedding of IL-1β–containing MPs. A-C show the percentage of (A) IL-1β–expressing platelets and (B) IL-1β–containing platelet-derived MPs and (C) the MFI of FLICA indicating caspase-1 activation in platelets isolated from healthy volunteers (control) or from DV-infected patients (dengue) that were positive (+) or negative (–) for signs of vascular permeability. The boxes indicate the median and interquartile ranges and the whiskers indicate 5 to 95 percentile. **P < .01 compared with healthy volunteers. ^#P < .05 between patients that were positive and negative for signs of increased vascular permeability. (D) Passive leakage of fluorescein isothiocyanate–conjugated albumin across endothelial cell layers was assessed after cell incubation with MPs recovered from mock- or DV-exposed platelets in the presence or absence of soluble IL-1Ra. The bars represent the mean ± SEM of 4 experiments using platelets from independent healthy donors. *P < .05 compared with mock MP-exposed endothelial cells layers. ^#P < .05 between endothelial cells layers that were incubated with sIL-1Ra or vehicle.

Figure 7

Schematic representation for DV-induced platelet shedding of IL-1β–containing MPs. Platelets synthesize IL-1β after DV exposure. In parallel, RIP1/RIP3-mediated mitochondrial ROS generation activates NLRP3, which recruits ASC and caspase-1 to assemble the inflammasome complex. Active inflammasomes cleave the full length IL-1β (p31) into the mature cytokine (p17) and promote the shedding of IL-1β MPs. Platelet-derived IL-β–containing MPs contribute to the increase in endothelium permeability.