Figure 4.

α-CD59 abolishes inhibitory effects of exosomes on C5b-9–induced NETosis and IL-17 production. (A) Representative images of NETs formation by neutrophils upon assembly of C5b-9, in the presence or absence of exosomes and α-CD59. Cells were stained for nuclei (DAPI, blue) and H3 Citrullinate R2/8/17 (green). Scale bar, 25 µm. (B) Quantification of NET release by neutrophils pooled from three independent experiments performed using neutrophils from four unique donors. At least 100 cells were quantified from each experiment. The neutrophils used in these experiments were from the same donors as those in Figures 1 and 2. Data shown as mean ± SEM (n = 4). *P = 0.02, two-way ANOVA. (C) mRNA expression levels of il17a relative to hprt1 in neutrophils upon assembly of C5b-9, in the presence or absence of exosomes and α-CD59 (n = 3). Three independent experiments were performed using neutrophils from three unique donors. The neutrophils used in these experiments were from the same donors as those in Figures 1 and 2. *P = 0.02, unpaired two-tailed Student's t test. ***P = 0.0003, one-way ANOVA. (D) C5b-9 were assembled on neutrophils in the presence or absence of exosomes and α-CD59, and culture supernatants were assayed for IL-17 by ELISA (n = 3). Three independent experiments were performed using neutrophils from three unique donors. The neutrophils used in these experiments were from the same donors as those in Figures 1 and 2. *P = 0.02, unpaired two-tailed Student's t test. **P = 0.004, one-way ANOVA. (Color version of figure is available online.)