Study On The Role Of High Mobility Group Box 1 Against Neutrophil Antibody - Induced Neutrophil Extracellular Network Formation

[Objective] The current study aimed to investigate whether HMGB1 participates in ANCA-induced neutrophil extracellular traps (NETs) formation, which is one of the most important pathogenic aspects in the development of AAV.[Methods] neutrophil isolation was obtained from healthy donors by venipuncture, NETs were induced by treating neutrophils with HMGB1 and ANCA-positive 1gG in vitro. The NETs were quantified by measuring cell free DNA (cfDNA) concentration with the Quant-iT Pico-Green fluorescence probe. We further measured the percentage of NETs formation by immunofluorescence.[Results] The typical components of NETs stained with DNA structures and colocalization of histone, granular proteins MPO was seen in HMGB1 plus with ANCA. The percentage of NETs formation was significantly higher in neutrophils stimulated with HMGB1 plus ANCA-positive 1gG than that in neutrophils incubated with HMGB1 or ANCA-positive 1gG alone. Compare with the non-stimulated neutrophils, HMGB1 or ANCA-positive 1gG alone, HMGB1 plus health 1gG, the cell free DNA concentration of NETs increased significantly n the neutrophils incubated with HMGB1 plus MPO-ANCA-positive IgG (286.60±29.58 ng/ml vs.513.97±116.29 ng/ml, P<0.001; 340.82±60.63 ng/ml vs.513.97±116.29ng/ml, P<0.001; 396.63±112.64 ng/ml vs.513.97±116.29 ng/ml, P=0.001,359.82±76.86 ng/ml vs.513.97±116.29 ng/ml, P=0.002, respectively). Consistently, Compare with the non-stimulated neutrophils, HMGB1 or ANCA-positive IgG alone, HMGB1 plus health IgG, the cell free DNA concentration of NETs increased significantly n the neutrophils incubated with HMGB1 plus MPO-ANCA-positive IgG(273.20±11.76 ng/ml vs. 540.25±130.95 ng/ml, P<0.001; 358.38±80.01 ng/ml vs.540.25±130.95 ng/ml, P<0.001; 334.78±87.16 ng/ml vs.540.25±130.95 ng/ml, P<0.001,407.25±89.90 ng/ml vs.540.25±130.95 ng/ml, P=0.018, respectively), No obvious NETs formation was observed with HMGB1, ANCA-IgG alone, normal IgG alone or HMGB1 plus normal IgG, either.[Conclusion] HMGB 1 can potentiate ANCA-inducing NETs formation and may be involved in the pathogenesis of AAV.[Objective] Since HMGB1 contributes to NETs formation in the presence of ANCA-positive 1gG, we next investigated whether TLR2, TLR4, RAGE and ROS were required in the process of HMGB1 promoting ANCA-induced NETs formation.[Methods] neutrophil isolation was obtained from healthy donors by venipuncture, NETs were induced by treating neutrophils with HMGB1 and ANCA-positive lgG in vitro. Certain groups of neutrophils were pretreated with blocking relevant antibodies before the incubating with HMGB1. The NETs were quantified by measuring cell free DNA (cfDNA) concentrationwith the Quant-iT Pico-Green fluorescence probe. We further measured thepercentage of NETs formation by immunofluorescence.[Results] In neutrophils incubated with HMGB1 plus MPO-ANCA-positive 1gG, the cell free DNA concentration was 537.25±90.11 ng/ml, which significant decreased compare with pre-incubating with anti-TLR2 antibody or anti-TLR4 antibody or RAGE antagonist or combined the three blocking antibodies and inhibitors (537.25±90.11 ng/ml vs.403.51±87.89 ng/ml, P=0.012; 537.25±90.11 ng/ml vs.386.18±97.14 ng/ml, P=0.003; 537.25±90.11 ng/ml vs.340.62±66.44 ng/ml, P<0.001; 537.25±90.11 ng/ml vs.371.89±70.22 ng/ml, P=0.001). ForPR3-ANCA-positive 1gG, the cell free DNA concentration was 540.33± 142.82 ng/ml, which significant decreased compare with pre-incubating with anti-TLR2 antibody or anti-TLR4 antibody or RAGE antagonist or combined the three blocking antibodies and inhibitors (540.33±142.82 ng/ml vs. 355.79±64.70 ng/ml, P=0.005; 540.33±142.82 ng/ml vs.367.42±73.51 ng/ml, P=0.009;540.33±142.82 ng/ml vs.371.46±56.36 ng/ml, P=0.008; 540.33±142.82 ng/ml vs.355.24±51.29 ng/ml, P=0.008). To further confirm the receptors through which HMGB1 exerts its effects, we used TLR2-/-and TLR4-/-mice. The aforementioned effect was significantly attenuated by blocking NADPH oxidase (533.66±118.10 ng/ml vs.458.33±136.59 ng/ml, P=0.010; 577.93±121.69 ng/ml vs.450.93±107.549 ng/ml, P=0.001).[Conclusion] HMGB1 exerts effects on NETs formation through interaction with TLR2, TLR4 and RAGE, and the process is NADPH-oxidase dependent.