BackgroundActivated platelets release cytokines/proteins including CXCL4 (PF4), CCL5 and fibrinopeptides, which regulate infection of several pathogenic viruses such as HIV, H1N1 and HCV in human. Since platelet activation is the hallmark of Dengue virus (DV) infection, we investigated the role of platelets in DV replication and also in a closely related Japanese Encephalitis virus (JEV).Methods and findingsMicroscopy and PCR analysis revealed a 4-fold increase in DV replication in primary monocytes or monocytic THP-1 cells in vitro upon incubation with either DV-activated platelets or supernatant from DV-activated platelets. The mass spectrometry based proteomic data from extra-nuclear fraction of above THP-1 lysate showed the crucial association of PF4 with enhanced DV replication. Our cytokine analysis and immunoblot assay showed significant inhibition of IFN-α production in monocytes via p38MAPK-STAT2-IRF9 axis. Blocking PF4 through antibodies or its receptor CXCR3 through inhibitor i.e. AMG487, significantly rescued production of IFN-α resulting in potent inhibition of DV replication in monocytes. Further, flow cytometry and ELISA data showed the direct correlation between elevated plasma PF4 with increased viral NS1 in circulating monocytes in febrile DV patients at day-3 of fever than day-9. Similarly, PF4 also showed direct effects in promoting the JEV replication in monocytes and microglia cells in vitro. The in vitro results were also validated in mice, where AMG487 treatment significantly improved the survival of JEV infected animals. Interpretation: Our study suggests that PF4-CXCR3-IFN axis is a potential target for developing treatment regimen against viral infections including JEV and DV.

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Neutrophils maintain immune homeostasis by engulfing apoptotic cells and debris. We describe the rapid activation of neutrophils after engulfing hemoglobin (Hb)-activated platelets, which are abundant in the circulation of hemolytic patients. Neutrophils from healthy individuals after engulfing Hb-activated platelets express elevated CD11b and secrete significant amounts of tumor necrosis factor (TNF)-α, interleukin (IL)-1β, IL-6, myeloperoxidase (MPO) and elastase within 4-h platelets, but not with free-Hb only in vitro. These neutrophils exhibit early onset of apoptosis and cell death after engulfing Hb-activated platelets, but not with free-Hb only. Further, our data from mice with phenylhydrazine-induced intravascular hemolysis display a gradual decrease in total neutrophil count, but the number of activated neutrophils and neutrophil-platelet aggregates increases, along with the rise of TNF-α, IL-1β, IL-6 and MPO in circulation. Our data from paroxysmal nocturnal hemoglobinuria (PNH) patients confirmed the observation of decreased total neutrophil counts, but elevated numbers of activated neutrophils, including neutrophil-platelet aggregates, in parallel with elevated expression of TNFA, IL1B and IL6 genes in neutrophils, also increased levels of these cytokines along with MPO in circulation, and this correlated directly with elevated intravascular hemolysis (high free-Hb in plasma). The patients' neutrophils displayed significant localization of intracellular Hb and platelets, unlike the counterparts from healthy individuals. Together, therefore, our observations suggest that Hbactivated platelets, which are abundant in the circulation of patients with hemolytic disorders, including PNH, promotes early onset of neutrophil activation and increases their proinflammatory response and leads to early apoptosis and cell death.