Fig. 2: Reduction in MPO-ANCA-IC-induced NET formation in neutrophils derived from MOD06051-administered normal rats.

Normal rats were administered orally bid with MOD06051 (3 mg/kg) or vehicle (0.5% methylcellulose) daily (n = 3/group). Two weeks later, neutrophils were separated from the blood using Polymorphprep and treated with 100 ng/ml rat TNF-α or 100 ng/ml mouse C5a for 15 min at 37 °C. Thereafter, these cells were incubated in MPO-ANCA-IC-immobilized 4-well slide chambers for 3 h at 37 °C. Cells were collected by pipetting in chambers and resuspended in EDTA-PBS were allowed to react with Sytox Green, and then submitted for FCM to detect Sytox Green⁺ NET-forming neutrophils. a The representative FCM profiles that reproduced in each group. b Comparison of NET-forming rate in TNF-α-primed neutrophils and C5a-primed neutrophils between MOD06051-administered rats and vehicle controls. Values are displayed as the mean with SEM, and p-values were calculated using a one-sided Student’s t test. c Neutrophils isolated from a non-treated normal rat were stimulated and harvested similarly and then subjected to fluorescent immunostaining for Cit-H3 and FCM using Sytox Green. Moreover, MPO-DNA complexes in supernatants were quantified by ELISA. *p < 0.05, **p < 0.01. Bar, 50 µm. Source data are provided as a Source Data file.