Figure 6: In vitro effect of 4-OI on cytokine release by human-monocyte derived primary macrophages and NET formation by human neutrophils.

A-E) Human primary monocyte-derived macrophages (at least n=6 from different donors) were obtained from healthy control or SLE subjects circulating monocytes by differentiating with either GM-CSF (GM proinflammatory) or M-CSF (M anti-inflammatory) for 7 days. Cytokines in supernatants were measured by ELISA after 24 h treatment with 4-OI or vehicle. The cytokines measured were A) IL1-beta, B) IL-6, C) TNF-alpha, D) IL-10, and E) IL-8. F) NET formation in normal dense granulocytes (NDGs) measured by fluorometry plate assay. Shown is mean ± SEM from SLE and healthy control (CTR) neutrophils, with numbers indicated in the figure, corresponding to 9 SLE and 3 healthy controls, done by technical duplicates; G-H) and by fluorescent microscopy imaging showing merged immunofluorescence staining with primary antibody against neutrophil elastase (green) and DNA (nuclei and NET fibers, Hoechst, blue); representative images of neutrophils from G) SLE and from H) healthy controls; magnification is 10 x. Bar graphs represent mean ± SEM. The statistical analysis was done using Mann Whitney test *:p<0.05; **:p<0.01