Figure 4.

Supernatants of monocytes and macrophages transfected with miR-4512 promote neutrophil extracellular traps formation. (A, B) Plasma levels of myeloperoxidase (MPO) (A) and cell-free DNA (cfDNA) (B) in SLE patients and control subjects from Tibetan and Han populations. Data are presented as the mean ± SEM. ****P < 0.0001, **P < 0.01, *P < 0.05. (C) Western blot analysis of neutrophil elastase (NE) expression in neutrophils isolated from SLE patients (A1–A4) and control subjects (B1–B4) from Tibetan and Han populations. Results are representative of three independent experiments, with actin used as an internal control. (D, E) MPO (D) and cfDNA (E) levels in the supernatants of human primary neutrophils culture stimulated with a supernatant of monocytes and macrophages transfected with NC/miR-4512-agomir/antagomir. Neutrophils directly stimulated with LPS or CXCL2 were used as positive controls. Data are presented as the mean ± SEM. ****P < 0.0001; ***P < 0.001; **P < 0.01; *P < 0.05; NS, not significant. (F) Images of neutrophils stimulated with macrophage and monocyte conditioned medium or with CXCL2 and LPS. Nuclei were stained with Hoechst (blue) and the presence of extracellular DNA were stained with Sytox Green. Images are representatives of three independent experiments. (G) Quantification of neutrophils extracellular DNA during NETosis by Sytox Green assays. Data are presented as the mean ± SEM. ****P < 0.0001; ***P < 0.001; *P < 0.05. (H) Western blot analysis of elastase expression in neutrophils after stimulation. Images are representatives of three independent experiments.