Immune Recognition of Citrullinated Cartilage PG Aggrecan in Autoimmune Arthritis

描述（由申请人提供）：蛋白聚糖（PG）聚集蛋白聚糖是关节软骨的主要大分子组分。PG被认为是类风湿性关节炎（RA）患者的T细胞自身抗原，类风湿性关节炎是一种外周关节的自身免疫性疾病，导致软骨的炎性破坏和关节功能丧失。PG的“致关节炎”潜力通过以下观察得到强调：用人PG免疫BALB/c小鼠诱导关节炎，其临床和免疫学特征类似于RA中的那些。我们最近发现从成人软骨中提取的PG是瓜氨酸化的。虽然瓜氨酸（精氨酸残基酶促转化为瓜氨酸）是一种翻译后蛋白质修饰，可能发生在健康个体中，但与健康受试者不同，大多数RA患者将瓜氨酸化自身蛋白识别为“外来”，并产生抗瓜氨酸化抗体。 蛋白自身抗体（ACPA）。重要的是，我们发现ACPA+ RA血清与体内或体外瓜氨酸化人PG反应，主要是与分子的第一个球状（G1）结构域反应。RA患者关节软骨中存在的瓜氨酸化PG有可能引发自身免疫反应和/或作为ACPA的靶点，并产生严重后果。然而，关于瓜氨酸化PG表位的T细胞识别的数据非常有限，并且对RA中PG特异性ACPA的表位库、软骨特异性沉积和致病性一无所知。目标1中描述的研究将（i）使用RA患者的外周血探索瓜氨酸化PG（G1结构域）特异性T细胞和ACPA表位库，以及（ii）鉴定沉积在受损RA关节软骨中的PG特异性ACPA。由于患有人PG诱导的或PG G1结构域诱导的关节炎（GIA）的小鼠也产生ACPA，因此该动物模型为研究瓜氨酸化PG特异性自身免疫的机制方面提供了独特的研究工具，而这在RA患者中无法研究。因此，在目标2和3中，我们将（i）在体外瓜氨酸化G1免疫的BALB/c小鼠中绘制人PG的G1结构域内的瓜氨酸化表位的完整库，（ii）监测瓜氨酸化G1特异性免疫的演变，（iii）将抗G1 ACPA的幅度和表位库与关节炎的严重程度相关联，和（iv）确定瓜氨酸化G1特异性免疫耐受的选择性丧失是否可导致关节炎的发展。我们的研究结果应提供瓜氨酸化PG在ACPA+ RA发病机制中的作用的临床相关见解。

DESCRIPTION (provided by applicant): Proteoglycan (PG) aggrecan is a major macromolecular component of articular cartilage. PG has been considered a T-cell autoantigen in patients with rheumatoid arthritis (RA), an autoimmune disease of the peripheral joints that results in inflammatory destruction of cartilage and loss of joint function. The "arthritogenic" potential of PG is underscored by the observation that immunization of BALB/c mice with human PG induces arthritis with clinical and immunological features similar to those in RA. We have recently found that PG extracted from adult human cartilage is citrullinated. While citrullination (enzymatic conversion of arginine residues to citrulline) is a post-translational protein modification that may occur in healthy individuals, the majority of patients with RA, unlike healthy subjects, recognize citrullinated self proteins as "foreign" and produce anti-citrullinated protein autoantibodies (ACPA). Importantly, we have found that ACPA+ RA sera react with in vivo or in vitro citrullinated human PG, predominantly with the first globular (G1) domain of the molecule. Citrullinated PG present in the joint cartilage of RA patients has the potential to initite autoimmune responses and/or serve as a target for ACPA with serious consequences. However, very limited data is available about T-cell recognition of citrullinated PG epitopes, and nothing is known about the epitope repertoire, cartilage-specific deposition, and pathogenicity of PG-specific ACPA in RA. The studies described in Aim 1 will (i) explore the citrullinated PG (G1 domain)-specific T-cell and ACPA epitope repertoire using peripheral blood from RA patients, and (ii) identify PG-specific ACPA deposited in cartilage of damaged RA joints. Since mice with human PG-induced or PG G1-domain induced arthritis (GIA) also produce ACPA, this animal model provides a unique research tool for investigating the mechanistic aspects of citrullinated PG-specific autoimmunity that cannot be studied in patients with RA. Therefore, in Aims 2 and 3, we will (i) map the full repertoire of citrullinated epitopes within the G1 domain of human PG in BALB/c mice immunized with in vitro citrullinated G1, (ii) monitor the evolution of citrullinate G1-specific immunity, (iii) correlate the magnitude and epitope repertoire of anti-G1 ACPA with the severity of arthritis, and (iv) determine whether selective loss of citrullinated G1-specific immune tolerance can lead to arthritis development. The results of our studies should provide clinically relevant insights into the role of citrullinated PG in the pathogenesis of ACPA+ RA.