IFN-λ1 (IL-29) inhibits autophagy to reduce neutrophil extracellular traps-associated thrombin production following stimulation with serum of patients with antiphospholipid syndrome / 中华微生物学和免疫学杂志

ABSTRACT

Objective:To investigate whether autophagy in the inflammatory environment of thrombotic antiphospholipid syndrome (APS) drives polymorphonuclear neutrophils (PMNs) to release neutrophil extracellular traps (NETs) and whether IFN-λ1(IL-29) has a function of inhibiting NETs-related thrombin production.Methods:PMNs isolated from peripheral blood of healthy volunteers were in vitro stimulated by serum samples of patients with active APS. IL-29 and 3-methyladenine (3-MA) were used to regulate NETs release and autophagy. NETs expression was evaluated by immunofluorescence technique (IFT) and flow cytometry (FCM). Autophagy-related proteins were detected by Western blot. The levels of thrombin-antithrombin (TAT) complexes were determined by enzyme-linked immunosorbent assay (ELISA). Results:The percentages of NETs released from PMNs of healthy volunteers after stimulation with serum samples of APS patients were (22.8±3.1)% according to IFT and (10.1±2.7)% according to FCM and used as control group. IL-29 and 3-MA inhibited the release of NETs from PMNs stimulated by serum samples of APS patients and the percentages of NETs were (5.3±2.2)% and (4.9±2.4)% detected by IFT, and (2.1±1.3)% and (2.7±1.4)% by FCM, respectively. There were significant differences with the control group ( q=9.89, 10.67, 11.74, 9.61, all P<0.01). IL-29 inhibited the expression of LC3B, but promoted the expression of p62 on PMNs of healthy volunteers after stimulating with serum samples of APS patients, and the differences with the group without IL-29 pretreatment were statistically significant [LC3B/GAPDH (0.28±0.03)% vs (0.77±0.06)%, q=8.65; p62/GAPDH (0.63±0.05)% vs (0.33±0.03)%, q=4.78; both P<0.01]. IL-29 and 3-MA reduced the levels of NETs-related TAT complexes from (7.6±0.6) ng/ml to (3.1±0.5) ng/ml and (4.7±0.4) ng/ml, respectively ( q=6.34 and 5.15, both P<0.01). Conclusions:Autophagy was involved in the formation of NETs and subsequent coagulation cascade activation in PMNs of healthy subjects after stimulation with serum samples of APS patients with thrombosis. IL-29 suppressed the production of NETs and NETs-associated thrombin by inhibiting autophagy.