William Harvey Research Institute, Queen Mary University of London, London, United Kingdom.
Department of Rheumatology, Royal Free NHS Foundation Trust, London, United Kingdom.
Front Immunol. 2023 Jun 2;14:1164941. doi: 10.3389/fimmu.2023.1164941. eCollection 2023.
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.
重度嗜酸性粒细胞性哮喘（SEA）的慢性气道炎症表明潜在的自身免疫发病机制，类似于抗中性粒细胞胞浆抗体（ANCA）阳性 EGPA（嗜酸性肉芽肿性多血管炎）中的髓过氧化物酶（MPO），存在未识别的自身抗体。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。SEA 中针对 oxPTM 自身抗原的自身抗体尚未进行研究。
招募 EGPA 和 SEA 患者以及健康对照参与者。抗原非特异性方法：将参与者的血清与未经刺激和 PMA 刺激的中性粒细胞和嗜酸性粒细胞载玻片孵育，并用抗人 IgG FITC 抗体通过免疫荧光鉴定针对粒细胞的自身抗体。靶自身抗原方法：从先前的文献和 FANTOM5 基因集分析中鉴定出嗜酸性粒细胞表达蛋白的候选蛋白。通过间接 ELISA 检测这些蛋白质的天然和 oxPTM 形式的血清 IgG 自身抗体。
免疫荧光研究表明，已知 ANCA 的患者血清预期会针对中性粒细胞 IgG 进行染色。此外，17 名 SEA 患者中有 9 名患者的血清针对 PMA 刺激的中性粒细胞进行 NETosis 进行了 IgG 染色。除了一名 SEA 个体表现出细微的核染色外，所有参与者（健康人和患有嗜酸性疾病的参与者）的血清对嗜酸性粒细胞载玻片的免疫荧光染色均明显呈弥漫性细胞质染色。FANTOM5 基因集分析确定触发受体表达在髓样细胞 1（TREM1）和白细胞介素 1 受体 2（IL1R2）作为除先前文献中鉴定的髓过氧化物酶（MPO）、嗜酸性过氧化物酶（EPX）和胶原蛋白-V 之外的嗜酸性粒细胞特异性靶标，以测试自身抗体反应。间接 ELISA 发现，SEA 患者血清中胶原蛋白-V、MPO 和 TREM1 的自身抗体浓度高于健康对照组。健康参与者和 SEA 参与者的血清中均可见高浓度的 EPX 血清自身抗体。与天然蛋白相比，在检查 oxPTM 时，自身抗体 ELISA 阳性患者的比例并未增加。
尽管研究的靶蛋白均没有对 SEA 具有高灵敏度，但至少有一种血清自身抗体阳性的患者比例很高，这表明对自身抗体血清学进行更多研究有可能改善严重哮喘的诊断检测。
ClinicalTrials.gov，标识符，NCT04671446。
