Fig 2: Role of platelet GM-CSF and C3 in neutrophil-DNA release. a–c Isolated human platelets and neutrophils were incubated together or by themselves for 30 min (at constant rotation and 37 °C) in the presence of TLR agonists [TLR7 (Loxo)—1 mM; TLR2 (Pam3CSK4, PAM)—10 µg/µL] or thrombin (IIa)—0.05 U/mL. GM-CSF release from a platelets (p = 0.8903, F = 0.2071, df = 3), b neutrophils (p = 0.7255, F = 0.4422, df = 3), and c platelets and neutrophils incubated together was measured by ELISA (p = 0.0200, F = 4.116, df = 3). The graphs represent the average fold change for each individual of n = 6 (3F; 3M) ± SD. d Confocal images of isolated human neutrophils treated with C3 (30 ng/mL) and neutrophils treated with C3 in the presence of GM-CSF (25 ng/mL). Neutrophils were treated in HEPES-modified Tyrode’s buffer (0.04 × 105 neutrophils/µL) for 30 min at 37 °C, and constant rotation (aggregometer). At the end, cells were fixed and stained [CD41-FITC-platelets (green); H4-AF637-histone (red); DAPI-DNA (blue)]. Neutrophil-DNA aggregates were visualized by confocal microscopy. Images are representatives of n = 4 different donors. C3-treated neutrophils from healthy donors release their DNA and form large aggregates. e Quantitation of (d). The graph (n = 4, 2F, 2M) is represented as average (p < 0.0001, F = 71.42, df = 3) ± SD. Significance in (a–c, e) was assessed using ANOVA followed by Bonferroni multiple comparison test and star symbol (*) indicates p < 0.05. Source data are provided as a Source Data file

Fig 3: Platelets contribute to C3 and Ly6G-DNA release in vivo. Platelets were eliminated from male mice with antiplatelet antibody CD42 (aPlt) and compared to control IgG. At 24 h post elimination, mice were infected with the PR8 strain of influenza (as in Fig. 8). a Confocal microscopy of blood showing DNA release in murine blood 3–4 days post-infection. Images are representative of n = 4 mice/group. b Quantitation of the confocal images in (a). Graph is a representative of 4 mice/group (p = 0.0003, F = 14.26, df = 3). c C3 levels in murine plasma at the same time as in (a). The graph represents the average levels ± SD, n = 4 mice/group, with the exception of IgG+sal, where n = 3 mice were used (p = 0.0415, F = 4.733, df = 3). Significance was measured by ANOVA followed by Bonferroni follow-up test; in all cases star symbol (*) indicates p < 0.05. d Gene expression levels of influenza RNA in isolated murine platelets 12 days post-infection (n = 4 of IgG+flu; n = 4 of aPlt+flu). The graph represents average expression ± SD; significance was calculated by two-tailed unpaired t-test, p = 0.0715, df = 6. Of note, Mann–Whitney non-parametric t-test gave p = 0.0286. Source data are provided as a Source Data file. Abbreviations: IgG—control antibody; aPlt—antiplatelet CD42b antibody; Sal—phosphate buffered saline: e Proposed mechanism of platelet-mediated neutrophil-DNA release during influenza infection. During influenza infection, virions cross into the circulation and become engulfed by platelets. Influenza virions lead to the release of complement C3 from platelets in a platelet-TLR7-dependent manner. C3 in turn activates neutrophils to release their DNA and leads to the formation of platelet–neutrophil aggregates that can circulate freely in blood. Aggregates of this nature can increase the risk for thrombosis and potentially lead to unstable coronary syndrome when there is vessel stenosis or inflamed endothelium

Fig 4: pDCs with Biallelic Expression Have Higher mRNA Levels of the Respective GenesComparison of mRNA expression levels for the five investigated genes TLR7, RPS6KA3, CYBB, BTK, and IL13RA1 between male monoallelic pDCs (yellow squares), female monoallelic pDCs (blue circles, monoallelic expression of the major allele; orange circles, monoallelic expression of the minor allele), and female biallelic pDCs (green circles), thus pDCs with escape from XCI in the respective locus. pDCs were sorted from 2-h CL097-stimulated PBMCs. For each gene, the cells from multiple individuals were combined. The male pDCs were derived from n = 3 males. Because heterozygosity of the respective SNPs was necessary for assessment of expression patterns, genes were assessed within different numbers of females: TLR7, n = 3 females; RPS6KA3, n = 3 females; CYBB, n = 2 females; BTK, n = 5 females; and IL13RA1, n = 2 females. Expression patterns were determined using the following SNPs: TLR7, rs3853839; RPS6KA3, rs7051161; CYBB, rs5964151; BTK, rs700; and IL13RA1, rs2495636. Median (red bars) with interquartile range (black bars) is shown. A mixed effects linear regression model with a random intercept was used to take into account intra-sample correlations. Only significant differences are displayed. *p < 0.05; **p < 0.01; ****p < 0.0001.

Fig 5: pDCs with Escape from XCI in TLR7 Transcribe Significantly Higher Levels of IFNa and IFNß mRNA(A and B) Comparison of mRNA expression levels of isolated, unstimulated pDCs for all IFNa subtypes and IFNß combined (A) and mRNA expression levels of isolated, 2-h CL097-stimulated pDCs for all IFNa subtypes and IFNß combined (B). Female monoallelic TLR7-expressing pDCs are displayed with blue circles (monoallelic expression of the major allele) or orange circles (monoallelic expression of the minor allele), and female biallelic TLR7-expressing pDCs are displayed with green circles (pDCs with escape from XCI in TLR7). Expression patterns were determined using the following TLR7 SNP: rs3853839. Individual pDCs from n = 3 females are displayed. Median (red bar) with interquartile range (black bars) is shown. A mixed effects linear regression model with a random intercept was used to take into account intra-sample correlations. ****p < 0.0001.(C) Comparison of mRNA expression of isolated, unstimulated pDCs of all IFNa subtypes and IFNß combined between the lowest quartile of TLR7-expressing pDCs with the highest quartile of TLR7-expressing pDCs. Female monoallelic TLR7-expressing pDCs are displayed with blue circles (monoallelic expression of the major allele) or orange circles (monoallelic expression of the minor allele), and female biallelic TLR7-expressing pDCs are displayed with green circles (pDCs with escape from XCI in TLR7). White circles are pDCs without any detectable mRNA for TLR7. Expression patterns were determined using the following TLR7 SNP: rs3853839. Individual pDCs from n = 3 females are displayed. Median (red bar) with interquartile range (black bars) is shown. A mixed effects linear regression model with a random intercept was used to take into account intra-sample correlations. ****p < 0.0001.(D) Comparing mRNA expression of pDCs of all IFNa subtypes and IFNß combined between female pDCs and male pDCs. pDCs were sorted from 2-h CL097-stimulated PBMCs. Expression patterns were determined using the following TLR7 SNP: rs3853839. Male pDCs (yellow squares), female monoallelic-TLR7-expressing pDCs (blue circles, monoallelic expression of the major allele; orange circles, monoallelic expression of the minor allele) and female biallelic-TLR7-expressing pDCs (green circles, pDCs with escape from XCI in TLR7). Expression patterns were determined using the following SNP: TLR7: rs3853839. Individual pDCs from n = 3 females and n = 3 males are displayed. Median (red bar) with interquartile range (black bars) is shown. A mixed effects linear regression model with a random intercept was used to take into account intra-sample correlations. *p < 0.05.(E) PBMCs of males (n = 7) and females (n = 7) were stimulated for 2 h with CL097. The collected supernatant was analyzed for panIFNa levels with an ELISA. Female results are shown independent of TLR7 expression pattern. Median with interquartile range is shown. Mann-Whitney test was used for statistical analysis. *p < 0.05.