严重嗜酸性粒细胞性哮喘(SEA)的慢性气道炎症提示潜在的自身免疫性病因,其自身抗体与ANCA阳性EGPA(嗜酸性肉芽肿伴多血管炎)中的髓过氧化物酶(MPO)相似。先前的研究表明,蛋白质的氧化翻译后修饰(oxPTM)是自身抗体反应可能逃避免疫耐受的重要机制。先前尚未研究SEA中针对oxPTM自身抗原的自身抗体。
■招募了EGPA和SEA患者以及健康对照参与者。自身抗原不可知方法:参与者血清与未刺激和PMA刺激的嗜中性粒细胞和嗜酸性粒细胞的载玻片一起孵育,用抗人IgGFITC抗体免疫荧光法鉴定粒细胞自身抗体。靶自身抗原方法:从先前的文献和嗜酸性粒细胞表达蛋白的FANTOM5基因集分析中鉴定出候选蛋白。这些蛋白质的血清IgG自身抗体,以原生和oxPTM形式,用间接ELISA检测。
■免疫荧光研究显示,如预期的,来自具有已知ANCA的患者的血清针对嗜中性粒细胞的IgG染色。此外,17名受测试的SEA患者中的9名血清对经历NETosis的PMA刺激的中性粒细胞进行IgG染色。嗜酸性粒细胞载玻片的免疫荧光染色在所有参与者(健康和患有嗜酸性粒细胞疾病)的血清中均有明显的弥漫性细胞质染色,除了一个SEA个体,其细微的核染色明显。FANTOM5基因集分析确定了TREM1(在骨髓细胞1上表达的触发受体)和IL-1受体2(IL1R2)作为嗜酸性粒细胞特异性靶标,以测试除MPO外的自身抗体反应,嗜酸性粒细胞过氧化物酶(EPX),和从以前的文献中鉴定的胶原蛋白-V。间接ELISA发现高浓度的血清自身抗体的胶原蛋白-V,MPO,和TREM1在SEA患者中的比例高于健康对照组。来自健康和SEA参与者的血清中都明显存在高浓度的针对EPX的血清自身抗体。与天然蛋白质相比,当检查oxPTM时,具有阳性自身抗体ELISA的患者的比例没有增加。
■尽管所研究的靶蛋白都没有表现出对SEA的高灵敏度,至少一种血清自身抗体阳性的患者比例较高,这表明更多的自身抗体血清学研究有可能改善重症哮喘的诊断测试.
■ClinicalTrials.gov,标识符,NCT04671446。
The chronic airway inflammation in severe eosinophilic asthma (SEA) suggests potential autoimmune aetiology with unidentified autoantibodies analogous to myeloperoxidase (MPO) in ANCA-positive EGPA (eosinophilic granulomatosis with polyangiitis). Previous research has shown that oxidative post-translational modification (oxPTM) of proteins is an important mechanism by which autoantibody responses may escape immune tolerance. Autoantibodies to oxPTM autoantigens in SEA have not previously been studied.
Patients with EGPA and SEA were recruited as well as healthy control participants. Autoantigen agnostic approach: Participant serum was incubated with slides of unstimulated and PMA-stimulated neutrophils and eosinophils, and autoantibodies to granulocytes were identified by immunofluorescence with anti-human IgG FITC antibody. Target autoantigen approach: Candidate proteins were identified from previous literature and FANTOM5 gene set analysis for eosinophil expressed proteins. Serum IgG autoantibodies to these proteins, in native and oxPTM form, were detected by indirect ELISA.
Immunofluorescence studies showed that serum from patients with known ANCA stained for IgG against neutrophils as expected. In addition, serum from 9 of 17 tested SEA patients stained for IgG to PMA-stimulated neutrophils undergoing NETosis. Immunofluorescent staining of eosinophil slides was evident with serum from all participants (healthy and with eosinophilic disease) with diffuse cytoplasmic staining except for one SEA individual in whom subtle nuclear staining was evident. FANTOM5 gene set analysis identified TREM1 (triggering receptor expressed on myeloid cells 1) and IL-1 receptor 2 (IL1R2) as eosinophil-specific targets to test for autoantibody responses in addition to MPO, eosinophil peroxidase (EPX), and Collagen-V identified from previous literature. Indirect ELISAs found high concentrations of serum autoantibodies to Collagen-V, MPO, and TREM1 in a higher proportion of SEA patients than healthy controls. High concentrations of serum autoantibodies to EPX were evident in serum from both healthy and SEA participants. The proportion of patients with positive autoantibody ELISAs was not increased when examining oxPTM compared to native proteins.
Although none of the target proteins studied showed high sensitivity for SEA, the high proportion of patients positive for at least one serum autoantibody shows the potential of more research on autoantibody serology to improve diagnostic testing for severe asthma.
ClinicalTrials.gov, identifier, NCT04671446.