[Abstract]
Objectives Adult-onset Still's disease (AOSD) is a severe auto-inflammatory disease. Neutrophil activation with enhanced neutrophil extracellular trap (NET) formation is involved in the pathogenesis of AOSD. Functional leukocyte immunoglobulin-like receptor A3 (LILRA3) has been reported to be associated with many autoimmune diseases. We aimed to investigate the association of LILRA3 with susceptibility and neutrophil activation in AOSD.
Methods The LILRA3 deletion polymorphism and its tagging SNP rs103294 were genotyped in 164 AOSD patients and 305 healthy controls (HCs). Impact of LILRA3 on clinical features and mRNA expression was evaluated. Plasma LILRA3 level was detected using ELISA and its correlation with disease activity and circulation NET-DNA level was investigated. LILRA3-induced NETs were determined using PicoGreen dsDNA dye and immunofluorescence in human neutrophils and neutrophil-like differentiated NB4 cell line transfected with LILRB2 siRNA.
Results We identified that functional LILRA3 was a risk factor in AOSD (11.0% vs. 5.6%, p=0.034, OR=2.089; 95% CI 1.030 to 4.291), and associated with leukocytosis (p=0.039) and neutrophilia (p=0.027). Functional LILRA3 mRNA expression was higher in LILRA3+/+ patients』 PBMC (p<0.0001) and neutrophils (p<0.001). Plasma LILRA3 level was elevated in AOSD (p<0.0001) and correlated with disease activity indicators and circulation NET-DNA complexes. Finally, enhanced NET formation was identified in neutrophils from HCs and inactive AOSD patients with LILRA3 stimulation, and impaired in LILRB2 gene knocked down NB4 cells.
Conclusions Our study provides the first evidence that functional LILRA3 is a novel genetic risk factor for AOSD, and functional LILRA3 may play a pathogenic role by inducing NETs formation.
【中文摘要】
目的：成人斯蒂爾病(AOSD)是一種嚴重的自身炎性疾病。中性粒細胞活化和中性粒細胞胞外捕獲網(NETs)參與AOSD的發病。功能性白細胞免疫球蛋白樣受體A3 (LILRA3)已被報導與多種自身免疫性疾病相關。本研究旨在探索LILRA3與AOSD的易感性及中性粒細胞激活的關係。
方法：對164例AOSD患者和305例健康對照(HCs)進行了LILRA3缺失多態性及其標記SNP rs103294的基因分型。評價LILRA3對臨床特徵和mRNA表達的影響。採用ELISA法檢測血漿LILRA3水平，探討其與疾病活動度以及與循環NET-DNA複合物水平的相關性。用PicoGreen dsDNA染色和免疫螢光檢測了LILRA3對於人外周血中性粒細胞、以及轉染了LILRB2 siRNA的NB4細胞系誘導而來的中性粒細胞樣細胞的NETs刺激作用。
結果：我們發現功能性LILRA3是AOSD的危險因素(11.0% vs. 5.6%， p=0.034, OR=2.089;95% CI 1.030 - 4.291)，並與白細胞增多(p=0.039)和中性粒細胞增多(p=0.027)相關。功能性LILRA3的mRNA表達在LILRA3+/+患者的PBMC (p<0.0001)和中性粒細胞(p<0.001)中較高。AOSD患者血漿LILRA3水平顯著升高(p<0.0001)，並與疾病活動指標和循環NET-DNA複合物相關。最後，在LILRA3刺激的HCs和非活動AOSD患者的中性粒細胞中發現NETs形成增強，而在LILRB2基因敲除NB4細胞中發現NETs形成受損。
結論：該研究首次證明了功能性LILRA3是AOSD的危險因素，而功能性LILRA3可能通過誘導中性粒細胞NETs形成而發揮致病作用。