Institute of Immunology, Faculty of Veterinary Medicine, and Center for Biotechnology and Biomedicine, Leipzig University, Leipzig, Germany.
Swiss Institute of Equine Medicine (ISME), Department of Clinical Veterinary Medicine, Vetsuisse Faculty, University of Bern, Bern, Switzerland.
Front Immunol. 2023 Dec 15;14:1293684. doi: 10.3389/fimmu.2023.1293684. eCollection 2023.
Severe equine asthma (SEA) is a common, chronic respiratory disease of horses characterized by hyperreactivity to hay dust which has many similarities to severe neutrophilic asthma in humans. SEA-provoking antigens have not been comprehensively characterized, but molds and mites have been suggested as relevant sources. Here, we identified relevant antigen candidates using immunoproteomics with IgG isotype-binding analyses.
Proteins from () were separated by two-dimensional gel electrophoresis followed by immunoblotting (2D immunoblots) resulting in a characteristic pattern of 440 spots. After serum incubation, antibody (Ig)-binding of all Ig (Pan-Ig) and IgG isotypes (type-2-associated IgG3/5, type-1-associated IgG4/7) was quantified per each spot and compared between asthmatic and healthy horses' sera (n=5 per group).
Ig binding differences were detected in 30 spots. Pan-Ig binding was higher with asthmatics compared to healthy horses' sera on four spots, and IgG3/5 binding was higher on 18 spots. Small IgG4/7 binding differences were detected on 10 spots with higher binding with asthmatics' sera on four but higher binding with healthy horses' sera on six spots. Proteins from the spots with group differences including mite and yeast proteins were identified by liquid chromatography mass spectrometry. The latter likely originated from the feeding substrate of the culture. Prioritized antigen candidates amongst the proteins identified were Der p 1, Der p 11, group 15 allergens, myosin heavy chain, and uncharacterized proteins. Additionally, yeast enolases, alcohol dehydrogenase (ADH), phosphoglycerate kinase (PGK), glyceraldehyde-3-phosphate dehydrogenase, and heat shock proteins were prioritized. Eleven antigen candidates were tested for confirmation by ELISAs using the respective proteins separately. Differences in asthmatics vs. healthy horses' serum Ig binding to Der p 1, Der p 18, and three yeast enzymes (enolase, ADH, and PGK) confirmed these as promising antigens of immune responses in SEA.
Antigens with relevance in SEA were newly identified by immunoproteomics, and yeast antigens were considered for SEA for the first time. Serum IgG3/5 binding to relevant antigens was increased in SEA and is a novel feature that points to increased type-2 responses in SEA but requires confirmation of the corresponding cellular responses.
严重马气喘（SEA）是一种常见的、慢性的马呼吸系统疾病，其特点是对干草粉尘的高反应性，这与人类严重中性粒细胞性哮喘有许多相似之处。SEA 诱发抗原尚未得到全面描述，但已提出霉菌和螨虫是相关来源。在这里，我们使用 IgG 同种型结合分析的免疫蛋白质组学来鉴定相关的抗原候选物。
从（）中分离蛋白质，然后通过二维凝胶电泳进行免疫印迹（2D 免疫印迹），得到 440 个斑点的特征模式。在血清孵育后，对每个斑点的所有 Ig（全 Ig）和 IgG 同种型（与 2 型相关的 IgG3/5、与 1 型相关的 IgG4/7）的 Ig 结合进行定量，并比较哮喘和健康马血清（每组 5 匹马）之间的差异。
在 30 个斑点中检测到 Ig 结合差异。与健康马血清相比，哮喘马血清中 Pan-Ig 结合在四个斑点上更高，而 IgG3/5 结合在 18 个斑点上更高。在 10 个斑点上检测到 IgG4/7 结合的微小差异，其中四个斑点上哮喘马血清的结合更高，六个斑点上健康马血清的结合更高。通过液相色谱质谱鉴定了具有组间差异的斑点中的蛋白质，包括螨和酵母蛋白。后者可能来自于培养物的饲料底物。在鉴定出的蛋白质中，优先考虑的抗原候选物包括 Der p 1、Der p 11、15 组过敏原、肌球蛋白重链和未鉴定的蛋白。此外，酵母烯醇酶、醇脱氢酶（ADH）、磷酸甘油酸激酶（PGK）、甘油醛-3-磷酸脱氢酶和热休克蛋白也被优先考虑。使用各自的蛋白质分别通过 ELISA 测试了 11 种候选抗原的确认。哮喘患者与健康马血清中 Ig 结合的差异表明 Der p 1、Der p 18 和三种酵母酶（烯醇酶、ADH 和 PGK）是 SEA 中免疫反应的有希望的抗原。
通过免疫蛋白质组学新鉴定了与 SEA 相关的抗原，并且首次考虑了酵母抗原。SEA 中相关抗原的血清 IgG3/5 结合增加，这是 SEA 中 2 型反应增加的新特征，但需要确认相应的细胞反应。
