Fig 2: Platelets Influence IL-1 Levels In Vivo(A) Representative FACS plots and quantification of platelets in the blood of mice treated with a platelet-depleting Ab (aCD42b) or IgG control.(B and C) IL-1ß levels in sera (B) or peritoneal lavage (C) from platelet-depleted mice after i.p. challenge with LPS + R848 (see STAR Methods).(D) FACS quantification of neutrophils and monocytes in peritoneal lavage of mice treated as in (C).(E) Correlation between plasma cytokines and blood cell counts or hemoglobin levels in naturally infected malaria patients (n = 78). Red lines, positive correlations; shaded areas, 95% confidence; squared symbols, outliers (see STAR Methods).(F) Volcano plot of log2 fold change versus significance (p value) highlighting the platelet-signature genes within transcripts of healthy (n = 14) versus pediatric patients (n = 22) with NLRP3 mutations (NLRP3mut) causing MWS/NOMID (Balow et al., 2013).See also Figure S3.

Fig 3: Platelets Enhance NLRP3 and Pro-IL-1 Transcription in Human Macrophages(A) Caspase-1 activity in cell-free supernatants of NLRP3-overexpressing mouse macrophages ± platelets (50:1 ratio). Cells were left resting (Unstim) or activated with Nigericin (10 µM, 90 min) without LPS priming.(B) qPCR analysis of the indicated genes in hMDMs ± platelet supernatants.(C) Confocal imaging and quantification of ASC specks in LPS-primed and Nigericin-activated hMDMs ± platelets (50:1 ratio). Scale bar, 11 µm.(D and E) Immunoblotting for IL-1ß (D) or caspase-1 (E) in cell-free supernatants, as well as ß-actin in cell lysates of resting, LPS-primed hMDMs ± platelets (50:1 ratio), or platelet supernatants. Different donors are indicated.(F) Caspase-1 activity in cell-free supernatants of hMDMs treated as in (C). Graphs show floating bars (with mean and minimum to maximum values) from pooled data from three or four independent experiments. Each symbol represents the average of technical triplicates from different donors.

Fig 4: Platelets Are Critical for the Production of IL-1 Cytokines by Human Primary Monocytes(A) Representative FACS dot plots of CD41 and CD14 expression in human PBMCs, standard (Std-Mo), or platelet-depleted (PD-Mo) CD14+ monocytes (see STAR Methods).(B) Quantification of platelets (CD41+CD14-), platelet-monocyte aggregates (CD41+CD14+), and platelet-free monocytes (CD41-CD14+) in PBMCs and isolated monocytes.(C) IL-1ß and TNF-a levels in cell-free supernatants of Std-Mo versus PD-Mo, or in platelet-depleted monocytes replenished with autologous platelets (PD-Mo + Plts, 50:1 Ptl:Cell), and stimulated as indicated.(D and E) IL-1ß levels released by non-canonically activated Std-Mo, PD-Mo, or PD-Mo + Plts, stimulated with LPS (1 µg mL-1) for 16 h (D) or by inflammasome-activated THP-1 s ± platelets (E). Data is presented as floating bars (with mean and minimum to maximum values) and pooled from independent experiments. Each symbol represents the average from technical triplicates per blood donor. Data in (D) shows bar graph with Mean + SD, from a representative of two independent experiments.See also Figure S2.

Fig 5: Candidalysin induces IL-1ß release in murine mononuclear cells. a IL-1ß release measured by ELISA in culture supernatants LPS-primed mBMDMs infected with C. albicans Wt, re-integrant (ece1?/? + ECE1) or mutant strains (ece1?/?, ece1?/? + ECE1?184–279) (MOI 6) or co-incubated with synthetic Candidalysin for 5 h. b Levels of bioactive IL-1 measured in culture supernatants of LPS-primed mBMDMs that were infected with C. albicans Wt, re-integrant (ece1?/? + ECE1) or mutant strains (ece1?/?, ece1?/? + ECE1?184–279) (MOI 6) or co-incubated with synthetic Candidalysin. Bioactive IL-1 was quantified by stimulation of EL4.NOB-1 cells culture supernatants and correlation of the secreted murine IL-2 to a concentration range of recombinant human IL-1ß. c The presence of processed IL-1ß (p17) detected by western blotting in the supernatant of LPS-primed or unprimed (no LPS) mBMDMs that were infected with C. albicans Wt re-integrant (ece1?/? + ECE1) or mutant strains (ece1?/?, ece1?/? + ECE1?184–279) (MOI 10) or co-incubated with synthetic Candidalysin for 5 h. A representative image of three independent experiments or donors is shown. d IL-1ß and e TNF levels measured by ELISA in culture supernatants of LPS-primed or unprimed mBMDCs respectively, that were infected with C. albicans Wt, re-integrant (ece1?/? + ECE1) or mutant strains (ece1?/?, ece1?/? + ECE1?184–279) (MOI 5) or co-incubated with synthetic Candidalysin for 5 h (mBMDMs) or 4 h (mBMDCs). Secreted IL-1ß (a, d) and TNF (e) were determined by ELISA. Values are represented as scatterplots and the median of at least three different replicates (n = 3). nd not detectable