PMC6583233_01
Clinical course of a patient.
fimmu-10-01334-g0001.jpg
PMC6583233_01_fimmu-10-01334-g0001.jpg
F1
He was treated with prednisolone with antibiotics for GNB infections and his clinical findings were recovered (Figure 1).
BPI-ANCAs induce NETs with hyper citrullinated histones. (A) Representative NETs staining of healthy or BPI-AAV patient IgGs-stimulated neutrophils using CitH3 (red) and DAPI (BLUE). (B) Representative Sytox-positive neutrophils live image. From the left, healthy IgGs (n = 3), MPO-AAV patient IgGs (n = 5), BPI-AAV patient IgGs. Right figure shows Sytox-positive areas quantified by Image J. (C) Representative CitH3-positive NETs and Sytox-positive neutrophils treated with TNFalpha, TNFalpha+mBPI-Ab, TNFalpha+mBPI-Ab+rBPI. Right figures show CitH3-positive area (upper) and Sytox-positive area (lower) under each situation. Data represent the mean+-SEM of three independent experiments and were analyzed using unpaired t-test (PRISM software, GraphPad). *p < 0.05. (D) BPI expression with or without TNFalpha stimulation. Green; BPI, BLUE; DAPI. (E) Sytox positivity of neutrophils treated with 3% serum, or 3% IgG depleted serum of patients with BPI-AAV, MPO-AAV (n = 3) and healthy control (n = 3). Scale bar in all figures, 50 mm. CitH3, citrullinated histone3; DAPI, 49,6-diamidin-2-phenylindol; BPI, bactericidal/permeability increasing protein; ANCA, anti neutrophil cytoplasmic antibody; AAV, ANCA associated vasculitis; IgG, immunoglobulin G; TNFalpha, tumor necrosis factor alpha; mBPI-Ab, monoclonal BPI antibody; rBPI, recombinant BPI.
fimmu-10-01334-g0002.jpg
PMC6583233_01_fimmu-10-01334-g0002.jpg
F2
Sytox Green-positive NET formation with histone citrullination under TNFalpha stimulation in a manner similar to that of MPO-AAV IgGs-treated neutrophils (Figures 2A,B). Since the polyclonal TNFalpha-dependent NET formation with hypercitrullination (Figure 2C). To elucidate the phenomenon that TNFalpha accelerated the ICs-induced NET formation, the expression of BPI in neutrophils with or without TNFalpha stimulation was evaluated by immunostaining. TNFalpha up-regulated BPI expression in neutrophils and the overexpressed BPIs were translocated to cell surface (Figure 2D).