Study On The Pathogenic Mechanism Of Neutrophil Extracellular Traps (NETs) In Pregnancy Complications Of Antiphospholipid Syndrom

Objective:Antiphospholipid syndrome(APS)is a group of autoimmune diseases mediated by anti-phospholipid antibodies(aPLs)with recurrent arteriovenous thrombosis and/or obstetric complications as the main clinical manifestation.aPLs/APS is one of the major causes of obstetric complications during pregnancy.The pathogenesis of APS-related obstetric complications is unclear and no effective treatment is available.The role of neutrophil extracellular traps(NETs)in the pathogenesis of autoimmune diseases has received much attention,but their role in the obstetric complications associated with APS is poorly known.We aimed to verify the relevance of NETs to APS obstetric complications,to investigate the pathogenic mechanisms of NETs in APS obstetric complications,and to explore the potential of NETs as a potential therapeutic drug target for APS obstetric complications through animal studies.Methods:In this study,we analyzed the ability of aPLs to stimulate neutrophils releasing NETsin vitro.In addition,we investigated the association of NETs with APS relatedobstetric complications by analyzing NETs levels in serum and NETs infiltration in placental villous in 66 APS patientswithobstetric complications,68 diseased controls with other causes of obstetric complications and 37 healthy controls.We further used HTR-8/Svneo human chorionic villus trophoblast cells to explore the effect of NETs on migration,inflammation and apoptosis of trophoblast cells.We also examined the co-localization of NETs with tissue factor(TF)or von Willebrand factor(vWF)in placental villous in APS patientswithobstetric complications.Finally,we used PAD4 knockout mice to establish a mouse model of obstetric complications induced by aPLs to investigate the effect of NETs deficiency on obstetric complications in mice.Results:Neutrophils from APS patients with obstetric complications were more susceptible tobeinduced releasingNETsbyionomycin,and both patient serum and purified IgG had the ability to strongly stimulate the release of NETs in vitro.serum levels of free DNA and MPO-DNA complexes were significantly higher in APS patients with obstetric complications than in disease controls(p<0.01)and healthy controls(p<0.01).Both DPI and anti-TLR4 antibodies partially reduced the release of NETs stimulated by aPLs.NETs treatment enhancedaPLs inducing TNF-αproductionby monocytes,and inhibited the migration of HTR8 cells.Co-culturing 1000ng/ml of NETs,which purified from APS patients with obstetric complications,with HTR8 cells for 48 h significantly promoted HTR8 cell apoptosis(p=0.02).Embryo absorption rate was lower in the model of obstetric complications established by aPLs induction in PAD4 knockout mice than in wild-type mice(8.78±4.51 vs 19.76±4.33),but not statistically different(p=0.17).Conclusions:aPLs may induce the release of NETs from neutrophils through both the NADPH-oxidase(Nox)-dependentpathway and TLR4-dependentpathway.NETs correlate with APS relatedobstetric complications.aPLs may be involved in the pathogenesis of APS relatedobstetric complications through procoagulation,promotion of trophoblast inflammation and apoptosis,and inhibition of migrationof trophoblast cells.NETs deficiencycausedbyPAD4 knockout couldbe protectiveinthe mouse model of obstetric complications established by aPLs induction.