目的:特发性炎性肌病(IIM)的诊断基于形态学特征和疾病相关蛋白的评估。然而,虽然应用广泛,各自的方法强加了实质性的偏见,观察员和个人染色方法。我们旨在使用自动形态测量方法来量化主要组织相容性复合物(MHC)-1,(MHC)-2和细胞间粘附分子(ICAM)-1的蛋白质水平,以减轻偏倚。
方法:对整个肌肉切片进行双重免疫荧光染色,以研究肌纤维和肌内膜血管中蛋白质表达的差异。我们分析了所有IIM亚型,包括皮肌炎(DM),抗合成酶综合征(ASYS),包涵体肌炎(IBM),免疫介导坏死性肌病(IMNM),异常铁蛋白病(DYSF),SARS-CoV-2感染和疫苗接种相关肌病。具有神经源性萎缩(NA)和正常形态的活检作为对照。对样品的子集进行大量RNA测序(RNA-Seq)。
结果:我们的研究强调了MHC-1,MHC-2和ICAM-1在诊断IIM亚型中的重要性,并揭示了不同的免疫学特征。RNASeq证实了我们方法的准确性,并确定了疾病亚型中的特定基因途径。值得注意的是,Asys,DM和SARS-CoV-2相关肌病显示肌内毛细血管ICAM-1表达增加,表明在这些情况下ICAM-1相关的血管激活。此外,ICAM-1在不同亚组之间表现出高度的区别,具有较高的敏感性和特异性。
结论:自动形态计量学分析提供了免疫相关蛋白的精确定量数据,这些数据可以整合到我们对IIM的病理生理学理解中。Further,ICAM-1对IIM病理的检测具有诊断价值。
OBJECTIVE: Diagnosis of idiopathic inflammatory myopathies (IIM) is based on morphological characteristics and the evaluation of disease-related proteins. However, although broadly applied, substantial bias is imposed by the respective methods, observers and individual staining approaches. We aimed to quantify the protein levels of major histocompatibility complex (MHC)-1, (MHC)-2 and intercellular adhesion molecule (ICAM)-1 using an automated morphometric method to mitigate bias.
METHODS: Double immunofluorescence staining was performed on whole muscle sections to study differences in protein expression in myofibre and endomysial vessels. We analysed all IIM subtypes including dermatomyositis (DM), anti-synthetase syndrome (ASyS), inclusion body myositis (IBM), immune-mediated-necrotising myopathy (IMNM), dysferlinopathy (DYSF), SARS-CoV-2 infection and vaccination-associated myopathy. Biopsies with neurogenic atrophy (NA) and normal morphology served as controls. Bulk RNA-Sequencing (RNA-Seq) was performed on a subset of samples.
RESULTS: Our study highlights the significance of MHC-1, MHC-2 and ICAM-1 in diagnosing IIM subtypes and reveals distinct immunological profiles. RNASeq confirmed the precision of our method and identified specific gene pathways in the disease subtypes. Notably, ASyS, DM and SARS-CoV-2-associated myopathy showed increased ICAM-1 expression in the endomysial capillaries, indicating ICAM-1-associated vascular activation in these conditions. In addition, ICAM-1 showed high discrimination between different subgroups with high sensitivity and specificity.
CONCLUSIONS: Automated morphometric analysis provides precise quantitative data on immune-associated proteins that can be integrated into our pathophysiological understanding of IIM. Further, ICAM-1 holds diagnostic value for the detection of IIM pathology.