IFN-λ1(IL-29)通过自噬抑制抗磷脂综合征血清刺激的中性粒细胞胞外诱捕网相关凝血酶的生成  

IFN-λ1(IL-29)inhibits autophagy to reduce neutrophil extracellular traps-associated thrombin production following stimulation with serum of patients with antiphospholipid syndrome

作　　者：俞秋兴[1] 杜鸿[1] 张海方[1] Yu Qiuxing;Du Hong;Zhang Haifang(Department of Laboratory Medicine,the Second Clinical Medical School of Soochow University,Suzhou 215004,China)

机构地区：[1]苏州大学第二临床医学院医学检验系,215004

出　　处：《中华微生物学和免疫学杂志》2020年第7期547-552,共6页Chinese Journal of Microbiology and Immunology

摘　　要：目的探讨抗磷脂综合征(APS)血栓形成的炎症环境中自噬是否参与多型核中性粒细胞(PMNs)释放中性粒细胞胞外诱捕网(NETs),以及IFN-λ1(IL-29)是否具有抑制NETs相关凝血酶生成的作用。方法体外研究活动期APS患者血清刺激健康志愿者外周血分离的PMNs,IL-29和3-甲基腺嘌呤(3-MA)作为NETs释放和自噬的调节因子,通过免疫荧光技术(IFT)和流式细胞术(FCM)评估NETs的表达,Western blot检测自噬相关蛋白的表达,酶联免疫吸附试验(ELISA)测定凝血酶-抗凝血酶(TAT)复合物水平。结果将APS患者血清刺激健康人PMNs释放的NETs作为对照组,其免疫荧光技术检测表达水平为(22.8±3.1)%,流式细胞术分析表达水平为(10.1±2.7)%。IL-29和3-MA抑制了APS患者血清刺激的健康人PMNs上NETs的释放,免疫荧光技术检测表达水平分别为(5.3±2.2)%和(4.9±2.4)%,流式细胞术分析的表达水平分别为(2.1±1.3)%和(2.7±1.4)%,与对照组相比差异均有统计学意义(q=9.89、10.67、11.74、9.61,P均<0.01)。IL-29抑制了APS患者血清刺激的健康人PMNs上LC3B的表达,促进了p62的表达,与APS患者血清刺激的健康人PMNs中自噬相关蛋白的表达水平相比,差异均有统计学意义[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;P均<0.01]。IL-29和3-MA降低了NETs相关TAT复合物的水平,与APS患者血清刺激健康人PMNs释放的NETs相关TAT复合物水平比较,差异均有统计学意义[(3.1±0.5)ng/ml和(4.7±0.4)ng/ml vs(7.6±0.6)ng/ml,q=6.34和5.15,P均<0.01]。结论自噬机制参与了APS血栓形成患者血清刺激的健康人PMNs中NETs的释放以及随后的凝血级联激活,IL-29通过抑制自噬减少NETs的生成以及其相关凝血酶的产生。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 produ

关 键 词：IL-29 自噬 NETS 凝血酶 

分 类 号：R392[医药卫生—免疫学]