Fig. 1. SP-D binds LPS (O128:B12) and suppresses LPS-induced NETosis.

a Dot blots showing that SP-D binds to LPS. LPS (O128:B12) at different concentrations was dotted onto a membrane, which was incubated with SP-D at different concentrations in the presence of 5 mM CaCl₂ or 20 mM EDTA. Human SP-D was obtained from the BAL of patients suffering from proteinosis. b–d NETosis kinetics in neutrophils isolated from healthy human peripheral blood, as assessed by SytoxGreen plate reader assays, where the %DNA released by the cells in the presence of 0.5% (v/v) Triton was considered as 100%. b LPS dose-dependently induces NETosis. LPS at concentrations of ≥5 μg/mL induced substantial amount of NETosis. c SP-D dose-dependently suppresses LPS (5 μg/mL)-induced NETosis (n = 3, the values shown are the average from three individual experiments performed on different days; Two-way ANOVA with Bonferroni’s multiple comparison post-test F(8,90) = 147.7, p < 0.0001; post-test *p < 0.05). d The differences in NETosis at 240-min time points shown in panel C (n = 3, the values shown are the average from three individual experiments performed on different days, One Way ANOVA with Tukey’s multiple comparison post-test, F(1.477,2.953) = 27.98, p = 0.0127; *p < 0.05; **p < 0.01). Error bars represent standard deviation. See Supplementary Fig. 4 for positive and negative controls for SytoxGreen Assay.