Role of peptidyl arginine deiminase 4 (PAD4) in anti-β 2GP1/β 2GP1 complex-mediated neutrophil extracellular trap formation / 中华微生物学和免疫学杂志

ABSTRACT

Objective:To investigate the role of peptidyl arginine deiminase 4 (PAD4) in anti-β 2GP1/β 2GP1 complex-induced formation of neutrophil extracellular trapping networks (NETs). Methods:Peripheral blood neutrophils were isolated from healthy humans by density gradient centrifugation. PAD4 expression was detected by Western blot after the neutrophils were incubated with anti-β 2GP1/β 2GP1 complex (100 μg/ml) for a certain period of time. PAD4 inhibitor Cl-amidine (10 μmol/L) was used to pretreat neutrophils. Changes in the expression of citrullinated histone 3 (CitH3) at protein level and the relative content of myeloperoxidase (MPO)-DNA were detected by Western blot and ELISA, respectively. A mouse thrombus model of antiphospholipid syndrome (APS) was established by inferior vena cava stenosis. Intervention experiments were performed by intraperitoneal injection of Cl-amidine (50 mg/kg). The expression of CitH3 at protein level in plasma was detected by Western blot. The concentration of circulating free DNA (cf-DNA) in plasma was measured with fluorescent staining. Thrombus in inferior vena cava was collected and weighted to evaluate whether inhibiting the activity of PAD4 would suppress the APS-IgG-induced formation of NETs and thrombosis. Differences among groups were analyzed by t test or one-way analysis of variance (ANOVA). Results:The expression of PAD4 induced by anti-β 2GP1/β 2GP1 complex was significantly down-regulated in the cytoplasm, but increased in the nucleus [(3.67±0.32) vs (1.47±0.19), t=10.22, P<0.05; (0.57±0.19) vs (2.97±0.31), t=11.49, P<0.05]. Cl-amidine significantly inhibited the anti-β 2GP1/β 2GP1 complex-induced expression of CitH3 protein by neutrophils [(2.46±0.47) vs (0.46±0.13), t=12.24, P<0.01], and reduced the MPO-DNA content in the culture supernatants [(4.09±0.94) vs (2.80±0.57), t=4.23, P<0.05]. In vivo, Cl-amidine significantly inhibited the expression of CitH3 protein [(3.97±0.56) vs (1.09±0.45), t=11.83, P<0.01] and decreased the content of cf-DNA [(2 685.0±735.8) vs (1 784.0±577.0), t=3.93, P<0.05] in plasma of APS mice. Compared with the experimental APS mice in the control group, the weight of thrombus in the APS mice pretreated with Cl-amidine was significantly reduced [(8.22±3.06) vs (4.89±1.90), t=2.27, P<0.05]. Conclusions:PAD4 was involved in the formation of NETs induced by anti-β 2GP1/β 2GP1 complex, which might play an important role in APS thrombosis.