Platelets Fuel the Inﬂammasome Activation of Innate Immune Cells

The inﬂammasomes control the bioactivity of pro-inﬂammatory cytokines of the interleukin (IL)-1 family. The inﬂammasome assembled by NLRP3 has been predominantly studied in homogeneous cell populations in vitro , neglecting the inﬂuence of cellular interactions that occur in vivo . Here, we show that platelets boost the inﬂammasome capacity of human macrophages and neutrophils and are critical for IL-1 production by monocytes. Platelets license NLRP3 transcription, thereby enhancing ASC oligomerization, caspase-1 activity, and IL-1 b secretion. Platelets inﬂuence IL-1 b production in vivo , and blood platelet counts correlate with plasmatic IL-1 b levels in malaria. Furthermore, we reveal an enriched platelet gene signature among the high-est-expressed transcripts in IL-1 b -driven autoinﬂammatory diseases. The platelet effect is independent of cell-to-cell contact, platelet-derived lipid mediators, purines, nucleic acids, and a host of platelet cytokines, and it involves the triggering of calcium-sensing receptors on macrophages. Hence, platelets provide an additional layer of regulation of inﬂammasomes and IL-1-driven inﬂammation. and 1% Sodium Pyruvate) complemented with 500 U ml -1 rhGM-CSF at a concentration of 2x10 6 ml -1 in 6-well plates to generate monocyte-derived macrophages. Cells were harvested at day 3, counted using a hemocytometer and seeded at a concentration of 1x10 5 /well in complete medium complemented with 125 U ml -1 rhGM-CSF in 96-well ﬂat-bottom plates and incubated overnight for experiments on the next day. human experiments) or 5x10 6 platelets ml -1 (for mouse BMDM experiments), in order to ﬁt the macrophage:platelets proportion of 1:50 (human) or 1:5 (mouse). For the generation of supernatants from the megakaryocytic cell line MEG-01, 1x10 6 cells ml -1 were used. Next, platelets or MEG-01 were left untreated (Unstim) or incubated at 37 (cid:4) C with LPS (200 ng ml -1 ) or thrombin (1.0 U ml -1 ) for 3 hours. After stimulation, the platelet cell suspension agent Lipofectamine 2000 in OptiMEM. After 15 minutes of incubation, the empty vesicles were combined in 1:1 proportion of 4 m g ml -1 Poly(dA:dT), and further incubated for 15 minutes. The dsDNA-containing vesicles were added directly on the cell culture for a ﬁnal volume of 100 m l of medium (ﬁnal stimulatory concentration of 1 m g ml -1 ). Cells were stimulated for 3 hours before cell-free supernatants were harvested for cytokine measurements. mixed with the cell-free supernatant and the luminescent signal was evaluated 30 minutes later. The extracellular lactate dehydrogenase (LDH) concentrations in cell-free supernatants were assessed using the Pierce LDH Cytotoxicity Assay, according to the manufacturer’s instructions. Samples were measured at an absorbance of 490 nm using a Spectra-max i3 plate reader (ThermoFisher). Control hMDMs cultured at the same conditions as the samples were lysed with the provided Lysis buffer (10x) and served as positive control for maximal LDH release. was determined to be 7 amino acids and carbamidomethylation at cysteine residues was considered as a ﬁxed modiﬁcation. Oxidation and acetylation were included as variable modiﬁcations. For the analysis, the match-between runs option was enabled.