Table_1_Scurfy Mice Develop Features of Connective Tissue Disease Overlap Syndrome and Mixed Connective Tissue Disease in the Absence of Regulatory T Cells.DOCX

Due to a missense mutation in the Foxp3 gene, scurfy mice are deficient in functional regulatory T cells (Treg). The consequent loss of peripheral tolerance manifests itself by fatal autoimmune mediated multi-organ disease. Previous studies have outlined the systemic inflammatory disease and demonstrated production of anti-nuclear antibodies (ANA) in scurfy mice. However, specific autoantibody targets remained to be defined. ANA are immunological markers for several connective tissue diseases (CTD) and target a large number of intracellular molecules. Therefore, we examined scurfy sera for the presence of different ANA specificities and further assessed the organ involvement in these animals. Indirect immunofluorescence was used as a screen for ANA in the sera of scurfy mice and dilutions of 1/100 were considered positive. Addressable laser bead immunoassays (ALBIA) were used to detect specific autoantibody targets. Subsequent histological tissue evaluation was verified by hematoxylin and eosin (H&E) staining. In our study, we observed that nearly all scurfy mice produced ANA. The most prevalent pattern in scurfy sera was nuclear coarse speckled, also known as the AC-5 pattern according to the International Consensus on ANA Patterns. U1-ribonucleoprotein (U1RNP) was found to be the most common target antigen recognized by autoantibodies in scurfy mice. Additionally, scurfy mice exhibited a mild myositis with histological characteristics similar to polymyositis/dermatomyositis. Myopathy-specific autoantibody profile revealed significantly increased levels of anti-SMN (survival of motor neuron) as well as anti-Gemin3 antibodies in scurfy sera. Overall, we demonstrate that the impaired peripheral tolerance in the absence of regulatory T cells in scurfy mice is associated with features of mixed connective tissue disease (MCTD). This includes, along with our previous findings, very high titers of anti-U1RNP antibodies and an inflammatory myopathy.

由于Foxp3基因发生错义突变，Scurfy小鼠（scurfy mice）缺乏功能性调节性T细胞（regulatory T cells, Treg），由此引发的外周耐受丧失表现为致死性自身免疫介导的多器官疾病。既往研究已明确该模型存在全身性炎症性疾病，并证实其可产生抗核抗体（anti-nuclear antibodies, ANA），但具体的自身抗体靶标仍有待明确。抗核抗体是多种结缔组织病（connective tissue diseases, CTD）的免疫学标志物，可靶向大量细胞内分子，因此本研究通过检测Scurfy小鼠血清中不同ANA特异性的存在情况，进一步评估了该模型小鼠的器官受累情况。本研究采用间接免疫荧光法对Scurfy小鼠血清中的ANA进行初筛，血清稀释度为1/100时判定为阳性；采用可寻址激光微球免疫测定（addressable laser bead immunoassays, ALBIA）检测特异性自身抗体靶标；后续通过苏木精-伊红（hematoxylin and eosin, H&E）染色验证组织学评估结果。本研究中，几乎所有Scurfy小鼠均可产生ANA，其血清中最常见的荧光模式为核粗斑点型，根据国际ANA模式共识（International Consensus on ANA Patterns），该模式也被称为AC-5型；研究发现，U1核糖核蛋白（U1-ribonucleoprotein, U1RNP）是Scurfy小鼠自身抗体识别的最常见靶抗原。此外，Scurfy小鼠可见轻度肌炎，其组织学特征与多发性肌炎/皮肌炎相似；肌病特异性自身抗体谱分析显示，Scurfy小鼠血清中抗运动神经元生存蛋白（survival of motor neuron, SMN）抗体及抗Gemin3抗体水平均显著升高。综上，本研究证实，Scurfy小鼠在缺乏调节性T细胞的情况下出现外周耐受受损，这与混合性结缔组织病（mixed connective tissue disease, MCTD）的特征相关，结合本团队既往研究结果，该特征具体表现为高滴度抗U1RNP抗体及炎症性肌病。