Ligation of SIRL-1 suppresses both spontaneous and anti-LL37-triggered NET release by SLE neutrophils.

<p>A) Healthy (HD) or SLE neutrophils were incubated with anti-LL37 antibodies with or without pretreatment with anti-SIRL-1 mAb. NET-DNA release was quantified after 3 h in multiple experiments by fluorescence microscopy to reflect the area covered by extracellular DNA (as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0078459#pone-0078459-g001" target="_blank">Figure 1B</a>). HD, mean±SEM of four independent donors is shown. **p=0.0014, paired Student’s <i>t</i> test. SLE, mean±SEM is shown (medium, n=11; anti-LL37, n=13). *p=0.0117; **p=0.0052, paired Student’s <i>t</i> test. B) Representative images of anti-LL37-stimulated SLE neutrophils with or without SIRL-1 ligation stained for extracellular DNA (Sytox Green, left panels) and total DNA (Hoechst 33342, right panels). Scale bars, 50 µm. C) Correlation of spontaneous and anti-LL37 antibody-induced NET formation by neutrophils from individuals with SLE. r²=0.4413; p=0.0258, Pearson’s correlation test. D) Flow cytometry analysis of SIRL-1 surface expression on freshly purified neutrophils from healthy donors and SLE patients. Representative histograms (grey filled, isotype-matched antibody; white dotted, anti-SIRL-1 mAb) are shown. E) Results from multiple independent donors (HD; n=6, SLE; n=7) are given. Average mean fluorescence intensity of each group±SD is indicated, Student’s <i>t</i> test.